®
OPERATOR’S MANUAL
BE3000
Immersible Optical Biomass Monitor
User manual for the following BugLab part numbers:
BE3200 probe
BE3100 base unit
Includes Instructions for BE3000 Virtual Instrument and
Data Viewing Software
BugLab LLC
www.buglab.com
[email protected]
Last updated: September 20, 2016
Notice
This publication and its contents are proprietary to BugLab LLC (“BugLab”), and are
intended solely for the contractual use of BugLab customers.
While reasonable efforts have been made to assure the accuracy of this manual, BugLab
shall not be liable for errors contained herein nor for incidental or consequential damage
in connection with the furnishing, performance, or use of this material.
BugLab reserves the right to revise this manual and make changes from time to time
without obligation by BugLab to notify any person of such revisions or changes.
BugLab does not assume any liability arising out of the application or use of any
products, circuits, or software described herein. Neither does it convey a license under its
patent rights nor the patent rights of others.
This publication and its contents may not be reproduced, copied, transmitted, or
distributed in any form, or by any means, radio, electronic, mechanical, photocopying,
scanning, facsimile, or otherwise, or for any other purpose, without the prior permission
of BugLab.
BugLab provides no warranties whatsoever used in connection with any BugLab device,
express or implied. Neither does it guarantee software compatibility with any off-theshelf software package or any software program that has not been written by BugLab.
Intended use of this system must be followed within the guidelines of this manual. In no
event will BugLab be liable for any damages caused, in whole or in part, by any
customer, or for any economic loss, physical injury, lost revenue, lost profits, lost savings
or other indirect, incidental, special or consequential damages incurred by any person,
even if BugLab has been advised of the possibility of such damages or claims.
The optical designs and circuit board designs in the BE3000 products are proprietary to
BugLab. The user may not copy any of the designs, either in whole or in part without
written permission from BugLab.
Windows is a registered trademark of Microsoft Corporation.
The BE3000 software is written in the LabVIEWTM development environment.
Copyright © 2016 National Instruments Corporation. All Rights Reserved.
Copyright © BugLab LLC 2016
All Rights Reserved
2
Cautions
CLASS 1 LASER PRODUCT
Complies with 21 CFR 1040.10 and 1043.11 except for deviations
pursuant to last Notice No. 50 dated 6/2007.
Do not drop or shake the probe or base unit.
The probe is designed to be immersed in aqueous solutions.
Do not expose to caustic chemicals.
The probe tip, probe connector, and base unit receptacle all
employ multi-mode fiber optics that must be kept clean and free from
scratches. When not in use, cover all of these apertures with the
supplied protective caps.
Before autoclaving, the plastic cap must be removed from the
probe, the probe must be disconnected from the base unit, and the
protective cap must be placed over the hybrid electro-optic probe
connector.
The base unit is not autoclavable. When disconnected from
the probe, place the protective cap over the base unit electro-optical
receptacle.
The low-voltage base unit is water-resistant, but it is not
water-proof. Keep the base unit, cables and power supply clean and
dry.
3
There are no user-serviceable parts inside the probe or the
base unit.
Do not expose the base unit to caustic chemicals or high
temperatures.
Do not leave fingerprints or dirt on the fiber optical interfaces.
Special cleaning devices are provided for cleaning the fiber optic
probe connector and base unit receptacle. The tip of the probe may
be cleaned with a light application of ethanol, methanol, or
isopropanol and gentle wiping with lint-free wipes. (Do not use
acetone to clean the probe. Some of the materials used in the probe
will rapidly degrade if exposed to acetone.)
Important note to BE3000 software users:
Before plugging the BE3000 into your computer,
make sure you have installed the software first.
Configure the power settings on your computer to
never go into sleep mode.
4
Table of Contents
A. Introduction
B. Principles of Operation
C. Getting Started
1. Unpacking the Instrument
2. System Requirements for Software Installation
3. Conventions and Shortcuts
4. Software Installation
5. Connecting the Probe and Base Unit
6. Configuring the Power Adapter
7. Setting Up and Configuring
8. Verification of Probe Performance
D. Setting up on a Bioreactor
1. Inserting the Probe
2. Preparing for Autoclaving
3. Setting the Baseline
E. Working with the Software
1. Modifying the Data Acquisition Window
2. Recording Events during Data Collection
3. Editing Annotations
4. Simultaneous Data Collection from Mult. Inst.
5. Terminating Data Collection
F. Biomass Calibration
1. Collecting Calibration Data
2. Editing, Generating, and Saving a Calibration
3. Running in Calibrated Mode
G. Data Viewer
1. Opening, Viewing, and Resaving Data Files
2. Retrospective Baseline Adjustment
3. Retrospective Calibration Adjustment
G. Working with the Instrument
1. Analog Output
2. Error Codes and Averaging
3. Warning Messages
4. Error Messages
5. Maintenance
6. Compliance Testing
End-User License Agreement
Appendix I. Instrument Specifications
Appendix II. Error Codes
Appendix III. BE2x00 Serial Protocol Specifications
Appendix IV. BE3000 Deviations from BE2x00 Serial Protocol
Appendix V. Analog Output Calibration Example
Appendix VI. Trouble-Shooting
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INTRODUCTION
This User Manual describes the operation of the BE3000 immersible
optical biomass instrument. The probe measures biomass in liquid
cultures using laser optical reflectance at 1310 nm. The optical reflectance
is measured from the tip of the probe out to a maximum distance of 3 cm.
The tip of the probe must always be positioned below the liquid-air
interface in order to provide accurate measurements. Biomass calibration
tools are provided so that results can be reported in any biomass units
desired, such as dry cell weight (g/L), optical density (OD), or cell density
(cells/mL). One or more factory default biomass calibrations (e.g.
Saccharomyces cerevisiae dry cell weight) are pre-programmed into the
instrument. Creating and storing additional biomass calibrations is easily
accomplished using the provided software. Tools are also provided for
baseline correction of media reflectance, as measured in the absence of
biomass.
Reading and collection of the instrument results can be accomplished in
three ways: (1) Through your bioreactor control software, (2) through the
analog output port, or (3) through the BE3000 Virtual Instrument
software. Options 2 and 3 are further described in this manual.
The BE3000 Virtual Instrument software communicates with up to six
BE3000 or BE2x00 instruments. In addition to data collection and
logging, the Virtual Instrument software provides tools for configuration,
calibration, and verification of instrument performance. At program startup, a search is automatically performed for all connected BE3000 probes
and BE2100 sensors. All configuration settings are displayed in tables,
and the settings can be modified with a simple mouse click. Data
acquisition is graphically displayed in separate tabbed windows for each
sensor. Important events that occur during the bioreactor run can be
marked directly on the graph, and are displayed in a summary table for
each sensor. A combined graph window also allows the results for all
sensors to be overlaid. A separate BE3000 Data Viewing program allows
previously acquired data to be viewed, manipulated, and resaved.
Detailed instructions for operating both the BE300 Virtual Instrument and
Data Viewing software are provided in this manual.
6
PRINCIPLES OF OPERATION
The BE3000 instrument employs a near infrared (1310 nm) laser to noninvasively measure back-scattering from biomass within liquid cultures.
The near infrared portion of the optical spectrum is invisible to the human
eye; you will not be able observe light emanating from the sensor. The
laser is directed from an optical fiber at the tip of the probe. Although the
BE3000 is classified as a Class 1 Laser Product that does not require
protective eyewear for operation, we recommend that you avoid staring
into the tip of the fiber optic cable when it is in operation. When the probe
is immersed in a liquid medium containing cell biomass, the laser light is
scattered by the cells or microorganisms, creating a “glow ball” of
monochromatic light. The intensity and size of the glow ball is dependent
on the biomass within the liquid culture. At early stages of growth, when
the biomass is low, the glow ball will be large in size and weak in
intensity. As the cells or microorganisms grow and divide, the density
will increase and the glow ball will reduce in size and increase in intensity.
Two optical fibers adjacent to the laser emission fiber at the probe tip are
used to collect back-scattered light from within the glow balls.
Although classical Optical Density (OD) measurements in a
spectrophotometer require dilution in order to accurately determine
biomass greater than about 0.5 g/L dry cell weight, the BE3000 instrument
is able to determine biomass from 0.01 to 200 g/L dry cell weight, without
dilution, without sampling, with one single sensor. The high sensitivity
and linearity of the BE3000 instrument allows the growth rate to be
accurately and rapidly assessed.
One of the advantages of using an infrared laser source for measuring cell
biomass is the avoidance of light absorbance by colored media
components (and colored vessel materials). This allows for the
measurement of true scattering rather than a combination of absorbance
and scattering. As a result, a highly linear relationship is maintained
between biomass concentration and the measured optical reflectance.
However, it is also important to realize that for biomass containing
strongly visible-light-absorbing chromophores (e.g. photosynthetic algae),
the chromophore absorbance may affect the agreement between a
conventional OD measurement and the result reported by the BE3000
instrument. The OD measured in the visible range by a
spectrophotometer will be influenced by both chromophore absorbance
and cell scattering, whereas the OD reported by the BE3000 instrument
will be based only on cell scattering. In such situations, if the relationship
between chromophore concentration and biomass is relatively fixed, it still
may be possible to generate a strong correlation between OD measured by
conventional methods and that reported by the BE3000 instrument.
However, it is important to be aware that changes in chromophore
concentration that are not accompanied by biomass change would not
7
affect the result reported by the BE3000 instrument, but would skew the
results determined by conventional visible spectrophotometry.
Another important consideration when comparing BE3000 instrument
results with conventional spectrophotometry, is the optical design of the
spectrophotometer. Most spectrophotometers are designed to make
accurate measurements of absorbance, but not scattering. A determination
of chromophore absorbance requires only a comparison of how much light
is extinguished within a sample when the chromophore is present at
different concentration levels (e.g. zero and a known concentration). By
contrast, in a scattering measurement, light is deviated from its path, rather
than being extinguished. As a result, the measured amount of scattering
will be dependent on the area and angle of scattered light that is captured
by the detection system. Since the detector size and geometric
arrangement is not standardized between different commercial
spectrophotometers, the Optical Density determined for biomass samples
can vary significantly (e.g. 50% or more variation between different
spectrophotometer models!).1 For this reason, if you would like the
BE3000 instrument to report results in OD units, it will be necessary to
calibrate your sensor to correlate with the specific spectrophotometer that
is used for the off-line OD measurement. A simple step-by-step guide for
generating a custom calibration for the BE3000 Instrument is described in
this manual (Biomass Calibration, Section G).
The relationship between back-scattered light intensity (as measured by
the BE3000 instrument) and biomass (such as dry cell weight) is weakly
dependent on the size of the scattering particles. For this reason, it is
recommended that separate calibrations be used for organisms with
grossly different cell sizes, such as Escherichia coli (typical cell diameter
0.5-1 m) and Saccharomyces cerevisiae (typical cell diameter: 5-10 m).
For mono-disperse cell cultures, these cell size differences can be
compensated using a single multiplicative factor (“calibration slope”).
Note that the 10-fold difference in cell diameters of these 2
microorganisms only has about a 2-fold effect on the BE3000 calibration
to biomass. For this reason, minor variations in cell diameter, such as are
observed in different stages of growth, or between different strains of the
same organism, will have a relatively minor effect on the BE3000
instrument accuracy. Note that correlation between biomass and OD
measurements performed using conventional spectrophotometry are also
cell-size dependent, and that this cell-size dependence is somewhat
different than for the BE3000 instrument, due to the difference between
the optical measurement geometries (e.g. transmission vs. reflectance).
More serious consideration must be given to organisms that do not grow
as mono-disperse cells (e.g. filamentous growth). The relationship
between OD (whether determined by conventional means or the BE3000
instrument) and biomass is non-linear for organisms that are not monodisperse in a liquid culture. As a result, OD can only be expected to
8
provide an accurate measure of biomass for mono-disperse liquid cultures.
For organisms that grow in clusters, OD can only be expected to provide a
qualitative estimate of biomass.
Correlation between biomass and BE3000 instrument measurements will
generally be highest during the exponential (aka “logarithmic”) phase of
cell growth. Cell lysis results in a dramatic change in the average particle
size. Once significant cell lysis has begun, such as typically occurs during
the stationary phase of cultures, there will no longer be a linear
relationship between biomass and optical measurements of scattering (by
either conventional spectrophotometry or by the BE3000 instrument).
Attempts to apply a non-linear fit to accommodate more than one particle
size at a time (such as happens due to cell lysis) is not likely to be reliable
because there is insufficient information to distinguish between changes in
particle size vs. changes in number of particles. For this reason, when
generating new calibrations, we recommend only using data collected
prior to the transition between exponential and stationary phases of cell
growth.
The measurement wavelength of 1310 nm was carefully selected to
balance optical penetration depth into the culture with measurement
sensitivity.2-4 Absorbance by water at 1310 nm limits the penetration
depth of light into aqueous solutions to a maximum of 3 cm. At shorter
wavelengths (i.e. towards the visible region of the spectrum) the
penetration depth increases. For example, at 850 nm, the penetration
depth can be more than 10 cm. Particularly in small vessels, this can make
it difficult to avoid interference from non-biological objects, such as
impellers, other probes, or the vessel wall. At longer wavelengths, the
penetration depth into water rapidly diminishes due to increasing
absorbance of light by water. For example, at 1450 nm, the penetration
depth is diminished to less than 1 mm. This has the effect of reducing the
effective measurement to such an extent that the sensitivity to cell biomass
is substantially diminished. At 1310 nm the measurement volume is small
enough so that measurements can be made in small vessels (e.g. as small
as 50 mL in a 250 mL shake flask), while at the same time maintaining a
very wide linear range of sensitivity to biomass (4 orders of magnitude).
Another long-known source of potential interference with optical
measurements of cell biomass is bubbles. For microbial cells grown in
bioreactors, very high gassing and stirring rates are often employed. By
using a small fiber optic probe the BE3000 measurement volume is
limited to approximately 200 L or less. The number of microbial cells in
this volume will be in the thousand to millions at the lowest concentrations
of interest, and will range up into the millions to billions at high
concentrations. As a result, the individual microbial cells that are inside
the optically sampled volume will change over time as the cells move
through the medium, but the mean number of cells will be nearly constant.
Bubbles are generally larger and less numerous than the cells, so the
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number of bubbles within the optically sampled volume will vary widely
as a function of time. By creating a 2 dimensional map of biomass as a
function of the reflectance distribution and central value, we have found
that the effects of changing bubbles and biomass can be effectively
separated.3,4 By applying this map to new measurements, accurate
biomass prediction is achieved over four orders of biomass magnitude,
despite widely varying agitation and sparging conditions.3,4
References
1. Myers et al. BMC biophysics 6.1 (2013): 4.
2. U.S. Patent 8,405,033. “Optical sensor for rapid determination of
particulate concentration”.
3. U.S. Patent Application 20150300938. “Particle Sensor with
Interferent Discrimination”.
4. International Patent Application PCT/US2015/026702. “Particle
Sensor with Interferent Discrimination”.
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GETTING STARTED
Unpacking the Instrument
The standard BE3000 instrument consists of:
Fiber optic probe with cable (2 m).
Protective cap for hybrid electro-optical connector.
C-clamp used to set the probe height within a bioreactor.
Hex key used to tighten/loosen the C-clamp.
Reflectance standards (“Low” and “High”).
BE3100 Base Unit.
Protective cap for hybrid electro-optical receptacle.
USB cable (2 m).
BE3000 Virtual Instrument and Data Viewing Software.
Additional components needed, if you will be using the analog output on
the base unit:
Power Adapter, 12 V, 1 A
Analog output cable (2 m) with lemo connector
Accessories:
Cleaning sticks for fiber optics in probe connector (box of 50)
Cleaning sticks for fiber optics in base unit recept. (box of 50)
Fiber optic cleaning solution (4 oz)
Optional accessories:
Extended (5 m) USB cable
One-click cleaning tool for fiber optics in probe connector
One-click cleaning tool for fiber optics in base unit receptacle
Intermediate range reflectance standards for linearity check
Note: Unpack and inspect all of the components to assure that they have
not been damaged in shipping.
11
System Requirements for Software Installation
Minimum System requirements:
1. Windows XP SP3 / Vista / 7 / 8 / 8.1 / 10 (32 or 64 bit) Operating
System
2. Minimum of 256 MB of RAM
3. Minimum 500 MB free hard disk space.
4. 1024 by 768 resolution (or higher) video adapter.
5. Microsoft-compatible mouse.
6. Available USB communications port.
The BE3000 software runs in the LabVIEWTM operating environment.
Two separate programs are provided: (1) the “BE3000 Virtual
Instrument”, and (2) the “BE3000 Data Viewer”. The Virtual Instrument
software gives you the ability to chart the progress of your fermentation in
real time, and annotate important events. Most importantly, the software
allows you to calibrate your BE3000 instrument to the units of your
choice. This calibration can then be written into instrument memory,
allowing the BE3000 instrument to run in calibrated mode without being
connected to a computer. The Data Viewer software allows you to open,
view, manipulate, and re-save data files that were previously acquired with
the Virtual Instrument software. The Data Viewer software does not
communicate with BE3000 instruments and can be run at the same time as
the Virtual Instrument software.
Conventions and Shortcuts
1. Bold text is used to indicate menu items and buttons that you
may select with your mouse, key combinations that you may
execute on your keyboard, and names of control and
indicators on the graphical user interface.
2. Italic text is used to indicate window names.
3. Bold italic text is used to indicate sections of this manual.
4. The » sign is used to indicate sub-levels of menu commands.
For example:
Start » Settings » Control Panel » Add/Remove
Programs
means; select the  Start menu, then select the
Settings sub-menu, further select the Control Panel
sub-menu, and then finally select the Add/Remove
Programs sub-menu.
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Software Installation
Note: Before plugging a BE3000 device into your
computer, make sure you have installed the software
first!
STEP 1: Insert the BE3000 software Compact Disk (CD) into
your computer.
STEP 2: If a Windows AutoPlay message pops up, under “Install
or run program from your media” choose “Run setup.exe”. If the
installation does not start automatically, browse the CD for the
“setup.exe” file and double-click on it. If Windows asks “Do you
want to allow the following program (setup.exe)…to make
changes to this computer”, click on the Yes button.
STEP 3: You will be prompted to choose destination directories
for both the BE3000 (BugLab) software and the LabView
(National Instruments) run-time engine used by the BE3000
software. Click on the browse buttons if you want to install these
programs somewhere other than the default directories shown.
Click on the Next button.
STEP 4: Review the software license agreements. If you agree,
then click on the “I accept…” buttons and then the Next button.
STEP 5: A summary of the software components that are about to
be installed is next displayed. Click on the Next button to begin
installation. Installation may take several minutes.
STEP 6: If the software has successfully been installed you
should see an Installation Complete window. Click on the Next
button.
STEP 7: The instrument driver (“BugLab BE3x00”) is next
installed. If a windows pop-up message asks whether you want to
allow changes to your computer, select “Yes”.
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Connecting the Probe and Base Unit
STEP 1: Remove the metal protective cap from the end of the
probe connector and from the mating receptacle on the BE3000
base unit. Line up the red line on the cable connector with the red
line on the base unit receptacle and insert the connector into the
receptacle until you feel it securely click into place. Connect the
two protective caps together so that they remain clean while not in
use.
STEP 2: Connect the provided USB cable to the base unit and to
the computer on which you have already installed the BE3000
software. The computer should automatically recognize the
BE3000 device and install the driver. Wait for the driver
installation to finish before launching the BE3000 software.
Note: if the device is not automatically recognized, it can be
installed manually by double-clicking on “BugLabBE3000.inf”,
which should be located in the following folder C:\Windows\Inf.
See the section USB Driver Trouble-Shooting at the end of
Appedix VI if you are still experiencing difficulties.
STEP 3 (OPTIONAL): If you will be using the analog output,
plug the provided power adapter into a power source and into the
base unit (if the plug adapter is not correct, first follow the
instructions in the following section for configuring the power
adapter with the plug adapter appropriate for your country). Plug
the analog output cable into the base unit and wire it to the device
on which it is to be read (e.g. analog input on a bioreactor
controller).
Configuring the Power Adapter for the BE3100
Base Unit (International Version only)
Note: Primary power is provided to the base unit via the USB
cable. Plugging in the power adapter is only necessary if the
analog output is to be used.
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International
Power Adapter
North American
Plug Adapter
Continental
Europe Plug
Adapter
UK / Ireland
Plug Adapter
Australia / New
Zealand Plug
Adapter
Plug Adapter
Release Switch
STEP 1: If there is already a Plug Adapter in the Power Adapter,
but it is not appropriate for your country, press the Plug Adapter
Release Switch, rotate the Plug Adapter counter-clockwise, and
remove the Plug Adapter.
STEP 2: Select the Plug Adapter appropriate for the country in
which the instrument is to be used, insert it into the Plug Adapter,
and rotate clockwise until it clicks in place.
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STEP 3: Check that the Plug Adapter is held securely in the
power adapter.
Setting Up and Configuring
STEP 1: Configure the power settings on your computer so that
sleep mode is disabled.
Step i: Select Start » Settings (in Windows 8, from the
desktop, simultaneously press the Windows and the “C”
key, and select “Settings”, and then “Control Panel”).
Step ii: Select System » Power & sleep.
Step iii: Choose “Never” for the Sleep setting.
Step iv: Press the “Save Changes” button, and then exit
out of the Control Panel window.
Note: Serial communication is interrupted when a
windows computer goes into “sleep” mode. This will cause
the BE3000 Virtual Instrument software to lose it’s
connection with the instrument.
STEP 2: Launch the BE3000 software:
In Windows 7 and earlier and Windows 10: select Start »
All Programs » BugLab » BE3000 » BE3000 Virtual
Instrument.
In Windows 8: Go to the start screen, right-click over an
empty space, and then select “All apps” (lower righthand corner of the screen). Scroll through the
applications until you find “BE3000 Virtual
Instrument”. Click once on the application.
STEP 3: When the program is started up, a search is
automatically initiated for all connected BE2x00 and BE3000
devices. For those BE2x00 and BE3000 devices that are
identified, all configuration settings are read from instrument
memory and put into program memory. This process may take
several minutes, but once complete, a list of all available devices
will be shown in the “BugEye Devices” page at the top of the
screen. All configuration settings, as read from instrument
memory, can also be viewed in the “Device Configuration” and
“Device Calibration” pages.
The bar separating the top and bottom portions of the window can
be adjusted (over a limited range) by clicking and holding the
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mouse on the bar and moving the bar to the new desired position
before releasing the mouse again. Notice that Sensor Tabs at the
bottom of the page are enabled according to the number of BE2100
and BE3000 instruments that were identified. The sensor number
on the sensor tabs corresponds to the “sensor #” columns in the
configuration tables at the top of the page. If you wish to change
the order in which the sensors are numbered you can do so by
entering the desired order into the “New Order” column of the
“BugEye Devices” page and then selecting the “Rearrange” button.
The sensor numbering will be automatically updated in all
configuration tables and in the Sensor Tabs at the bottom of the
page.
STEP 4: Select the “Software Settings” tab at the top of the page.
The “Sampling Interval” column in the table determines the
frequency with which data is read from the sensor, displayed in
graphical form, and saved to file (data is always saved to file as
soon as it is acquired). The default setting for the Sampling
Interval is 60 seconds. If you wish to change the sampling
interval, type in a new value (in units of seconds). Notice that the
sampling interval is individually configurable for all available
sensors.
STEP 5A: Select the “Device Configuration” tab at the top of the
page. The table column labeled “Ave. Time (2 min)” determines
the averaging time constant setting for the BE3000 instrument.
The averaging time constant determines how quickly the
instrument responds to change. The larger the time constant the
smoother the data and the slower the response. The default setting
is 2 minutes. To set the averaging time to a new value, click on the
current value and select from among the choices (ranging from 0
seconds to 8 minutes). Note that as soon as you select a new value,
it is written into probe memory. The new value will persist across
power cycling, and will automatically be loaded into the table if
you restart the program. The settings are individually configurable
for BE3200 probe.
STEP 5B (OPTIONAL): The Device Configuration Table column
labeled “Growth Window (8 min)” determines the time window
over which the determination of growth rate is performed. The
growth rate is determined from a linear fit to the natural logarithm
of the biomass vs. time. During exponential growth the slope of
this fit corresponds to the growth rate of the organism (provided in
units of inverse hours). The Growth Window setting determines
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the time window over which the fit is performed. If you plan to
use this feature and want to change the Growth Window setting,
click on the current setting and select from among the allowed
settings (ranging from 1 to 32 minutes).
STEP 5C (OPTIONAL): The Device Configuration Table
columns labeled “Base Corr. (Off)” and “Base. Val. (0.0)”
determine respectively whether baseline correction is applied to the
data and the value of this correction. Under normal operation it is
recommended that the baseline correction be determined after
initiating data collection but prior to inoculation (see the section
D.3 below: Setting the Baseline).
STEP 5D (OPTIONAL): The Device Configuration Table
columns labeled “Biomass Cal. (Off)” determines whether a
calibration is used to transform the optical reflectance data into
biomass. Up to 10 factory default biomass calibration and up to 10
additional user-defined biomass calibrations can be stored in
instrument memory. To select a pre-defined biomass calibration,
under “Cal. Type” select “Factory Default”, and then under “Cal.
#” scroll through the available calibration (1-10). Not all 10
Factory Default biomass calibrations may be defined, in which
case the “Calibration Name/Units” field will display “Unused”. If
the pre-defined biomass calibrations do not include your organism
and desired reference method then you may wish to generate a
custom biomass calibration. Step-by-step procedures for
generating a new calibration are described in section F Biomass
Calibration. If none of the pre-defined calibrations are suitable
and you have not already generated a custom calibration, set
Biomass Cal. to Off (if it is currently On). If you have found a
suitable pre-defined calibration or have already generated a custom
calibration and wish to now apply it to the new data you are about
to collect, select the “Device Calibration” tab near the top of the
screen and check that the calibration coefficients and units have
been properly set. If the settings are not correct, follow the steps in
the Calibration section of this manual. Once the calibration
coefficients and units are properly set, return to the Device
Configuration table and set Biomass Cal. to On.
18
Verification of Instrument Performance
Before using the BE3000 instrument to measure biomass in a
liquid culture you may wish to verify that it is performing as
expected. This step is recommended when you are unpacking and
using the instrument for the first time, but may also be used to
periodically check sensor functionality. In addition to providing a
verification of performance, the option of recalibrating the sensor
is also provided. When swapping a probe between different base
units, the sensor check procedure should be used to account for any
differences across base units.
The following procedure describes how to run the “Sensor Check”
function via the Virtual Instrument Software.
STEP 1: Remove the protective cap from the tip of the probe and
check that the tip of the probe is clean. The probe tip may be
cleaned with alcohol using lint-free tissue. DO NOT USE
ACETONE, as it may cause irreparable damage to components
used in the probe.
STEP 2: The “low reflectance standard” is a vial filled with
distilled water. If the vial is new, remove the protective seal, and
fill it with clean distilled water. If the vial is not new, replace the
water in the vial with clean distilled water. Note that the plastic
reducing aperture on the low reflectance standard is removable, to
make it easier to replace the water. Completely fill the vial and
replace the plastic insert. Place the probe just inside low
reflectance standard, so that it is immersed in the liquid and at least
3 cm away from the bottom of the vial. If necessary, tilt the probe
and vial in order to keep any bubbles away from the tip of the
probe:
STEP 3: Start the “Check Sensor” function.
If you have not done so already, follow the steps under
Software Installation, earlier in this manual.
Step i Start up the software and wait for the initial Device
Search to complete. In the lower half of the screen, select
the tab of the sensor for which you wish to run the Sensor
19
Check function. Press the “Check Sensor” button (in the
lower left of the screen).
Note: The Sensor Check function cannot be run
while data acquisition is active.
Step ii: Once the probe has been properly inserted into the
low reflectance standard (see step 2, above), press the
button labeled “Start Sensor Check”, and then press “Start
Low Refl Stnd Meas”.
Step iii: The instrument will make 25 measurements
(lasting about 1 second each) on the low reflectance
standard. The standard deviation of the 25 measurements is
used to assess stability. If the measurements were unstable,
the message “The Low Refl Stnd measurement was
unstable.” will be displayed. If this occurs, make sure that
the tip the probe is clean and free from bubbles and that the
water in the low reflectance standard is clean. You will not
be allowed to proceed to measurements with the high
reflectance standard measurements until stable
measurements on the low reflectance standard have been
collected.
Step iv: Once stable measurements on the low reflectance
standard have been collected, the control button will read
“Start High Refl Stnd Meas”. The high reflectance
standard consists of silicon dioxide micro-beads suspended
in distilled water with a preservative (0.005% Thimerosal).
Check the expiration date on the bottle, and if necessary,
replace it (available through BugLab LLC). Thoroughly
mix the contents of the high reflectance standard by
shaking and/or vortexing until no sediment can be seen on
the sides or bottom of the tube, and then shake/mix for
several additional seconds. Note that the contents of the
high reflectance standard settle very quickly and must be
fully mixed immediately prior to the start of the high
reflectance standard measurement. Remove the probe from
the low reflectance standard, dry the tip with a lint-free
tissue, and then insert it into the high reflectance standard
in the same manner as it was inserted into the low
reflectance standard. Press the button labeled “Start High
Refl Stnd Measurement”, then the “OK” button in the popup screen that appears.
Step v: 25 readings will now be collected on the high
reflectance standard. If the readings were unstable, the
message “The High Refl Stnd measurement was unstable”
will be displayed. If this occurs, make sure that the tip of
the probe is clean and free of bubbles and that the high
reflectance standard is fully mixed. It will be necessary to
repeat the low reflectance standard measurement (step ii,
above) before proceeding again with the high reflectance
20
standard measurement. If the readings were stable, a
“Pass/Fail” assessment of sensor performance will be
made. This Pass / Fail assessment is based on a comparison
of the present measurements to prior measurements made
on the reflectance standards during manufacture.
STEP 4: Whether Pass or Fail is indicated at the end of the reflectance
standard measurements, you will be given the option of updating the
sensor calibration coefficients based on the measurements just completed.
If the “Set New Coeff” option is selected, new sensor coefficients will be
written into sensor memory. When swapping a probe to a new base unit,
the “Set New Coeff” option should always be selected. The new
coefficients are determined by linearizing the just-completed reflectance
standard measurements to the original reflectance standard measurements
collected at the time of probe manufacture. These new sensor coefficients
will persist even across power cycling of the instrument.
SETTING UP ON A BIOREACTOR
Inserting the Probe
The BE3200 probe can be adapted to fit into many different types
of bioreactors. Most commonly the probe is inserted through a
compression fitting in the head plate of the bioreactor. Note that
compression fitting are not suitable for high pressure applications.
Three examples are provided below:
The probe is provided with a small protective cap attached
to the probe tip. Make sure to remove this cap prior to inserting
the probe into the vessel head plate. Save the protective cap for
later use.
3 mm diameter probe version
STEP 1: The 1/8” (3.2 mm) diameter version of the probe fits
directly into an M8 port of an Applikon Mini-Bioreactor. The
same type of adapter (Applikon part number V3MP070171) that
holds a sparge tube can be used to hold and secure the BE3000
probe. This adapter consists of an O-ring that sits in the bottom of
the M8 port and a stainless steel hollow screw that screws into the
M8 port. When the hollow screw is tightened it compresses the Oring around the probe, thereby securing it. Loosen the hollow
screw within the port before inserting the probe. If you have
21
trouble inserting the probe, fully remove the hollow screw from the
M8 port and insert it onto the probe first, then push the probe
through the O-ring. Reinsert and screw the hollow screw into the
M8 port but do not fully tighten it yet.
STEP 2: Using the supplied hex key, loosen the metal clamp at
the top of the probe. Position the probe so that the tip is at least 1
cm below the media level and 3 cm from the bottom of the vessel
(or any other interfering objects). The ideal position for the probe
is in a region of high flow, such as adjacent to an agitator, in order
to prevent the accumulation of bubbles or debris on the probe tip
during the run.
STEP 3: Tighten the hollow screw into the M8 port using the
special tool that Applikon supplied with the Mini-Bioreactor.
Verify that the probe is secure. Tighten the metal clamp at the top
of the probe, using the Allen key.
4 mm diameter probe version
Coming soon!
Pg13.5 diameter probe version
Coming soon!
Please contact BugLab at (925) 208-1952 or [email protected] for
further advice if you are having trouble.
Preparing for Autoclaving
Disconnect the hybrid electrical/fiber optic connector from the
base unit and secure the protective caps over both the probe
connector and the base unit receptacle. Coil the fiber optic cable
into an autoclave bag before inserting the bioreactor into the
autoclave.
Initiating Data Collection
STEP 1: After auto-claving and cooling, but prior to inoculation,
clean the fiber optic interfaces on the probe and base unit (see
22
Maintenance), then re-connect the probe to the base unit and start
the BE3000 Virtual Instrument software. Select the Sensor Tab
(lower part of the window) corresponding to the sensor number for
which you want to begin data collection. Press the Start button.
STEP 2: You will be prompted to enter a file name for your data.
A default filename is automatically created, which consists of the
year, month, day, hour, minute, and second at which you pressed
the Start button. Choose the directory where you wish to store the
data and then modify the file name as desired and press OK. The
*.bug extension is added automatically. Data will automatically be
saved to this file as soon as it is transmitted from the BE3000
device.
If you selected a file name that already exists you will have the
options of choosing another file name, overwriting the existing
file, or appending to the existing file. Note that selecting
Overwrite will result in the old data file(s) being deleted, so
choose this option carefully!
Selecting Append allows you to continue a previously aborted
experiment. If you choose this option, the old data will be loaded
and the new data will be added to it. The time expired between the
earlier and the current experiment will be automatically accounted
for. Appending is only allowed if the serial number of the
currently selected BE3000 probe matches that in the file header of
the previously written data file. Also, all settings are read from the
old data and event file and used to update the sensor configuration
before the appended data acquisition commences.
Selecting Change will return you to the Select a filename for data
storage Window.
STEP 3: At this point, you are now actively collecting data and it
will begin to appear on the graph window for the selected sensor
number. New data points will appear at the time interval you
previously chose (see Setting up and configuring), so do not be
alarmed if you do not immediately see new data points appearing
on the graph. When data collection is started, the “Biomass
Readings” table is automatically selected in the top portion of the
program window. Each time a new reading is taken by the sensor
this table is updated with the new time-stamp and biomass reading.
The biomass reading displayed in this table is processed according
to the configuration settings; if baseline correction is on, then this
23
result will be baseline corrected; and if user calibration is on, the
biomass will be reported in user calibrated units. A numerical
Error Code and its interpretation are also displayed in the table.
An error code of 0 and the error code interpretation “Normal
Operation” are displayed when the sensor is operating in the
normal range. If the sensor is operating outside of its normal
range, warning or error messages will be displayed. See Sections
G.2-G.4 for further description of conditions under which warnings
and errors may be displayed. For a complete listing of the warning
or error messages that may be displayed see Appendix II. Error
and Warning Code. The Display Mode selector to the upper left
of the graph allows you to select the data that is displayed on the
graph (either Raw, Baseline Corr., Calibrated or Growth Rate).
Setting the Baseline
Baseline correction provides a means of subtracting off signals emanating
from reflectance sources in the bioreactor that are not of interest. For
example, in a typical application, the baseline will be measured just prior
to inoculation, thereby subtracting off the contribution of media
constituents from the reported results. This function is similar to
“zeroing” of a spectrophotometer using only medium prior to performing
an OD measurement.
Before collecting a new baseline it is important to establish that the signal
is stable. Viewing the signal in graphical format, such as provided in the
Virtual Instrument software can be helpful for this purpose. If the baseline
appears to be wandering excessively, it can sometimes be helpful to
temporarily reduce the sensor averaging time constant to “0” while
adjusting the probe position and/or the bioreactor conditions.
In the following, it is assumed that the probe has already been inserted into
the bioreactor, and that a stable reading has been achieved.
To set the baseline, the user normally specifies a range (start and end
point, as selected by right mouse button), and the baseline is determined
by averaging over the specified range. Alternatively, the baseline can also
be set manually. The following step-by-step description shows how to use
either method.
STEP 1: While running an experiment, the Baseline can be set by
positioning the mouse arrow over the graph window at the time
position at which you would like to start baseline averaging and
clicking on the right mouse button. From the drop-down lists that
appear choose “Create Annotation” >> “Baseline Start”.
24
STEP 2: The Add Annotation Window now becomes active.
Under the box labeled “Positioning Method:” notice that the
selected method is “Cursor”. This means that the Baseline Start
will be marked at the position at which you right-clicked the
mouse. Alternatively, if you wish to start the baseline averaging at
the most recently collected data point, select “Add to End”. Click
on the “Add Annotation” button at the bottom left of the pop-up
window.
STEP 3: Notice that a red marker and number have been added
onto the graph, indicating the start of baseline averaging. An
“event” has also been added to the “Event List” located to the left
of the graph. Continue the data acquisition process until you
would like to define the end point for baseline determination.
Position the mouse arrow over the graph window at the time
position at which you would like to end baseline averaging and
clicking on the right mouse button. From the drop-down list that
appears select “Create Annotation” >> “Baseline End”.
STEP 4: The Add Annotation Window again becomes active.
Leave the “Positioning Method:” set to “Cursor Position” if wish
to mark the “Baseline End” at the point where the mouse was
right-clicked. Alternatively, if you wish to start the baseline
averaging at the most recently collected data point, select “Add to
End”. Click on the “Add Annotation” button at the bottom left of
the pop-up window.
STEP 5: You have now defined the start and end points for
baseline determination, so you are ready to set the baseline. Right
click the mouse button anywhere over the graph. From the dropdown list that appears select “Create Annotation” >> “Baseline
Set”.
STEP 6: The Set Baseline Interactively Window now appears.
When this window is brought up, the current baseline value is
automatically read from the sensor and is displayed in the
“Baseline Value” box. To define a new baseline based on the start
and end points you have just selected, choose “Compute from
graph” in the box labeled “Baseline Method”. The newly
computed value is displayed in the “Baseline Value” box.
Alternatively, if you wished to set the baseline manually, you
could have selected “Set manually” as the “Baseline Method”.
Then, you could manually enter a new value into the “Baseline
Value” box. Note that when the “Set manually” option is selected,
the current sensor reading is written into the Baseline Value
25
window, but you may edit this value, if desired. Select the “OK”
button at the bottom left of the pop-up window.
You have now set a new baseline value. The time at which the new
baseline value was set is recorded and displayed in the text box in the
main window. In addition, the annotation is numbered and marked on the
graph. Notice that the baseline is applied only prospectively (data points
that were acquired prior to setting the baseline are not affected by the new
baseline).
When a new baseline is set via this “create annotations” method, the
baseline correction is automatically turned On. Baseline correction can
also be manually turned On or Off within the Device Configuration table.
Modifying the Data Acquisition Window
Several options are available to allow you to customize the manner in
which the data is displayed in the Data Acquisition Window.
1. You can change the scaling of the graph axes:
By right-clicking over the graph, the autoscaling of both the
X and Y axes can be turned on or off. When auto-scaling is
turned on the BE3000 software will select axis limits that
best display all of the data collected since the data acquisition
was begun. When autoscaling is turned off, you can change
the values on the axes by clicking one or more of the extreme
values of the grid labels and changing their values. The pan
(depicted as a hand) and zoom (depicted as a magnifying
glass) features at the left bottom of the graph can also be used
to change the range of the graph that is displayed. If you pan
or zoom while autoscaling is on, the graph will rescale
whenever new data is added to the graph that falls outside of
the current window. Regardless of the mode selected, all of
the data will always be stored to your selected data file.
Note: autoscaling can also be turned on and off by clicking
on the lock symbol within the scale legend.
2. You can use the cursor to read the value of a specific data
point:
First, select the cursor tool, which is depicted with cross-hairs
and is located to the bottom left of the graph. Next, use the
cursor positioning tool (four diamond shapes located at the
bottom center of the graph) to move the cursor to the desired
location. The cursor can also be moved with the mouse, by
left-clicking the mouse over the cursor, and dragging the
26
cursor to a new location whiling keeping the mouse button
depressed. The X and Y values of the data point where the
cursor is located are indicated within the cursor box (at the
bottom, right of the graph).
By right mouse-clicking over the cursor box, several other
options can be accessed. The cursor can be centered within
the screen by selecting the “Bring to Center” option.
Additional cursors can also be created or deleted.
3. You can change the displayed data type by selecting the
switch at the upper left of the graph to Raw, Baseline Corr.,
Calibrated, Growth Rate, or Error Code. Note that the
unless otherwise selected, the displayed data type will be set
according to the Device Configuration table (if User Cal. is
On, then the data type will be Calibrated; if User Cal. is Off
and Baseline Corr. is On, then the data type will be Baseline
Corr.; if both User. Cal. and Base. Corr. are Off, then the
data type will be Raw). By selecting the Growth Rate data
type you can observe a real-time estimate of the exponential
growth rate of your organism (in units of inverse hours). By
selecting the Error Code data type, you can quickly identify
whether any data points were collected under conditions of
error (negative error codes), warning (positive error codes),
or normal operation (error code = 0).
Note: In all of the display views, data points that
were collected under normal conditions are
displayed in light green. Data points collected
under warning or error conditions are depicted in
yellow or red, respectively.
4. By right-clicking on the data markers within the plot legend
(to the upper right of the graph) you can change many aspects
of the graph including the plot style, marker color, and
marker symbol.
Recording Events during Data Collection
A helpful tool provided in the data acquisition graph windows is
annotation (or event marking). As you alter conditions during a bioreactor
run, you can easily note them, and they will be automatically timestamped.
Note: Data acquisition must be started before the create annotation
feature becomes active in the individual sensor graphs.
27
STEP 1: To record an event, simply right-click on the area of the
plot where you wish to record an annotation, and select “Create
Annotation”.
Several pre-defined event types are available, including:
- Inoculation
- Calibration Sample Removal
- Sparge Rate Change
- Agitation Rate Change
- Foam Breaker Rate Change
- pH Change
- Temperature Change
- Nutrient Addition
- Induction
- Harvesting
- Baseline Start
- Baseline End
- Baseline Set
- Delete All Annotations
You also can enter a User-Defined event for non-standard events.
STEP 2: Select one of the event labels from the list.
STEP 3: In the Add Annotation window, various options can be
set and recorded depending on what event is selected. Additional
comments can be added within the text box labeled “Edit the
annotation text below, if desired”. A “Value” and “Units”
associated with the event can also be entered. For example, if the
agitation rate was set to 500 rpm, 500 could be entered into the
“Value” box and “rpm” could be entered into the “Units” box.
The positioning of the annotation on the graph is determined by the
“Positioning Method” selector box. The annotation can be added
to the end of the dataset (corresponding to the moment in time
when “Add Annotation” was initiated), or it can be added at a
manually selected point in time (time can be specified under the
“Graph Position” label), or it can be added at the cursor position
where the mouse was clicked.
STEP 4: When all parameters have been satisfactorily edited,
click on the Add Annotation button. The annotation is numbered
and marked on the graph, as well as being displayed in a text box
to the left of the graph, for future reference.
28
Note: Like the sensor data, the event data is saved to file as soon as it is
generated. The filename into which the event data is saved is the same as
the sensor data file except the extension is “*.evt” instead of “*.bug”.
Editing Annotations
Annotations can be edited by double-clicking on cells within the event
table and typing in a new value or text string. Modifications made to the
event table are saved to file as soon as you finish typing them in and hit
the enter key, or exit the table cell you were editing.
Events may also be deleted by right-clicking the mouse over an
annotation, and selecting “Delete Annotation”. This action will result in
removal of the annotation marker from the graph, deletion of the event
from the event table, and deletion of the event from the saved event file.
Simultaneous Data Collection from Multiple
Instruments
Following the same procedures described above, data acquisition can be
initiated on up to 6 BE3000 instruments simultaneously. If a new BE3000
device has been connected since the last search was performed, select the
“Search Again” button within the BugEye Device window (top portion of
the screen). Performing a new search will not interrupt data acquisition on
devices that are already active.
Data from multiple sensors can be overlaid and viewed by selecting the
“All Sensors” tab. The data type that is displayed for each sensor on the
“All Sensors” graph is determined by the data type that was selected on
the individual sensor plots. Thus, if the “Calibrated” data type was
selected in the “Sensor 1” graph window, the data that will be displayed
for Sensor 1 in the “All Sensors” window, will also be “Calibrated”. The
sensors that are displayed in the “All Sensors” graph can be controlled by
using the “On/Off” column in the “Multi-Plot Settings” Table. The
relative positioning of the different sensors can be editing the “X Offset”,
“Y Offset”, and “Y Scaling” values in the “Multi-Plot Settings” Table.
Annotations can also be added to the “All Sensors” graph. However, these
annotations are only saved in temporary memory and are not written to
file. In order to save annotations into permanent record, they must be
marked on the individual sensor graphs.
Terminating Data Collection
29
Selecting STOP terminates data collection.
30
BIOMASS CALIBRATION
If you wish to convert the BE3000 reflectance data into reference units
other than reflectance, you will need to apply a biomass calibration. One
or more factory default cell biomass calibrations are programmed into the
instrument at the time of manufacture (e.g. dry cell weight of
Saccharomyces cerevisiae). Your BE3000 software can also help you to
collect and then apply a custom biomass calibration file. When applying
your biomass calibration you will no longer need to perform aliquot
extraction (or any other classical method) in order to determine biomass or
related quantities, as the BE3000 sensor will now do that for you.
Collecting Biomass Calibration Data
This section describes how to collect a custom biomass calibration file for
your BE3000 instrument. For most accurate results, we recommend that
you collect a custom biomass calibration for your particular organism and
reference method (e.g. OD(600) on a particular spectrophotometer).
STEP 1: Run a fermentation as you normally would with the
BE3000 probe properly inserted and monitoring. Follow the
directions in the earlier section of this manual entitled Initiating
Data Collection.
STEP 2: At representative points during the fermentation, collect
calibration samples. Right-click over the graph each time a
calibration sample is removed. You will note a special Annotation
type called Calibration Sample Removal. Each time you record a
Calibration Sample Removal Event, the software records the
time, the event number, and the raw sensor output. Note that the
off-line reference value and units can also be recorded now in the
“Value” and “Units” fields. However, more commonly, the offline values will be entered at some later time, as they become
available.
Click on the “Add Annotation” button. Notice that calibration
events are marked on the graph in blue. Use the event number to
keep track of the samples you collect for off-line analysis.
STEP 3: Repeat step 2 until a full calibration set has been
collected. The calibration file must contain a minimum of one
point, but we strongly suggest more. It is recommended that
calibration points spanning the lowest and highest biomass are
recorded. Collection of calibration points during the exponential
31
growth phase (prior to stationary phase) usually results in the most
successful calibration.
STEP 4: Press STOP when a full calibration set has been
collected.
Editing, Generating, and Saving a Calibration
STEP 1: Once you have completed a fermentation run during
which you collected calibration samples, select Cal Window
within the sensor graph window for which you want to generate a
calibration. The User Calibration Window appears. The data
table on the left side of the screen is automatically populated with
the calibration samples from the Event List currently loaded. If
you wish to read in the calibration samples from a different file,
select “Read from Event File”. Alternatively, if you wish to bring
up a previously saved calibration file, select “Read from Cal File”.
STEP 2: The data table contains four columns: “Event #”, “Raw
Sensor”, “Baseline”, and “Calibration”. The “Sensor” column
contains the data reported by the BE3000 instrument at the time at
which the calibration sample was extracted. The “Baseline”
column contains the baseline value that was used to collect the
BE3000 data. The “Calibration” column contains the matching
off-line reference data that will be used to generate the calibration.
If you have not entered any calibration data yet, the Calibration
value will be -1. Add the off-line reference values to the
Calibration column. Make sure that the “Event #” corresponds to
the correct sample. The data displayed on the graph will be
updated as soon as you enter it into the table.
STEP 3: If necessary, adjust the Raw Sensor and Calibration
measurement offsets by using the Adjust Baseline, Fixed
Intercept?, and Intercept controls. The value in the Baseline
column of the calibration table is subtracted from the Raw Sensor
readings before correlating them to the Calibration values. By
pressing the Adjust Baseline button, you can simultaneously
change the Baseline setting for all measurements in the Calibration
Table. Individual Baseline values can be adjusted by directly
editing the values in the Baseline column of the Calibration Table.
When the Fixed Intercept? control is checked, the linear fit is
forced to intersect with the y-Intercept at a BE3000 reflectance
reading of zero. It is recommended that you keep the Fixed
32
Intercept? control checked, unless you have entered Reference
values that span both the low and high range of the biomass units
into which you are calibrating. When the Intercept is fixed,
generally it is recommended to keep it fixed at zero, unless the
reference method has a built-in offset that you haven’t already
accounted for (e.g. if you measured OD in a complex medium and
didn’t zero the spectrophotometer using the media alone, then you
should enter the OD of the medium alone as the Intercept value).
STEP 4: Enter the type of reference data into the “Calibration
Units” box at the top left of the graph. It is recommended that you
include means of identifying both the organism and the reference
method (e.g. “e coli g/L”). But be aware that if you type in a name
that is longer than 31 characters (including spaces), it will
automatically be truncated to 31 characters.
STEP 5: Choose the data transform method. Generally the
“Linear-Linear” is recommended. If your samples were collected
at logarithmic intervals, the “Log-Log” transform may work best.
However, be aware that an error will be reported if the baselinecorrected reflectance value is less than or equal to zero.
STEP 6: Choose the Polynomial order that will be used to fit the
data. We recommend using the lowest polynomial order that
adequately fits the data. Due to the high linearity of the BE3000
sensor response to biomass, a linear fit (polynomial order = 1) is
generally recommended. The root mean-squared (RMS) error and
linear correlation coefficient (R2) for the fit are shown below the
graph. RMS Error indicates the root mean squared difference
between the linear fit and the actual data, so the smaller the RMSE,
the better the fit. R2 values can range between 0 and 1, with 1
indicating perfect linear correlation. The polynomial coefficients
(“Poly Coeff”) resulting from the fit are also displayed below the
graph. The coefficients are listed from lowest to highest
polynomial order: offset, linear, quadratic, and cubic.
STEP 6: If you are satisfied with the fit, select the “Accept Fit”
button. You will be prompted to choose a path and name for the
calibration file to be saved. Following file saving, the new
calibration is automatically written into instrument memory.
Biomass calibration coefficients are stored in the base unit memory
and will be remembered across power cycles.
33
Running in Biomass Calibrated Mode
Now that you have saved a biomass calibration into sensor memory, if you
wish to collect biomass calibrated data, you simply need to turn On
biomass calibration. Calibration can be turned On within the Virtual
Instrument software from the “Biomass Cal.” column of the Device
Configuration table (tab at the top of the screen).
Note: When running in biomass calibrated mode, the “Error Code
Interpretation” column of the “Biomass Readings” table will display a
warning message if the raw sensor data falls outside the calibrated range.
In the event of this condition, the message “Warning: Extrapolating
Beyond Calibration” will be displayed.
DATA VIEWER
Opening, Viewing, and Resaving Data Files
A separate “BE3000 Data Viewer” program is provided for displaying
previously acquired data. The overall organization and appearance of the
program is much like that of the “BE3000 Virtual Instrument”. Previously
acquired files are opened (using the “Open” button) from within the “File”
tabs, located in the bottom half of the program window. Files can be resaved (using the “Resave” button), but a new filename must first be
selected. A suggested filename is automatically generated, that consists of
the original filename appended with the date and time at which it was
resaved. Resaving with the original filename is disabled in order to
protect against accidental overwriting of data. This is particularly
important when the Data Viewer program is used to open a file into which
data is still actively being acquired (by the “Virtual Instrument” program).
The data that is displayed in the Data Viewer program is a copy of the data
that was present at the time the file was opened (the viewed data file is not
automatically updated as new data points are collected). As with the
Virtual Instrument program, the last tab in the files tabs contains a graph
in which data from multiple files can be overlaid.
The configuration tabs in the top half of the Data Viewer screen are also
similar to those within the Virtual Instrument program. However, in the
Data Viewer, the configuration settings cannot be modified (“read-only”).
These configuration settings are as read from the header of the data files
that have been opened.
34
Retrospective Baseline Adjustment
The Data Viewer program also provides the capability to retrospectively
adjust the baseline that is applied to the data. This feature is useful when
you want to apply the same baseline setting across the entire file. If the
baseline was changed one or more times during data acquisition (with the
Virtual Instrument program), the changes were applied prospectively. In
such cases, different segments of data have different baseline settings.
The retrospective baseline adjustment feature allows you to equalize the
baseline setting across all data segments.
STEP 1: From within one of the “File” tabs, press the “Open”
button, select the file you want to work on, and then press “OK”.
STEP 2: From within the same “File” tab, press the “Adjust
Base.” button. The “Set Baseline Retrospectively.vi” window will
open.
STEP 3: Set the “Baseline Method” control to one of the three
settings:
“Set manually”: If you select this option, you should type
the new baseline value directly into the “Baseline Value”
control.
“Retrieve from config. table”: If you select this option,
the value that is in the “Device Configuration” table (in the
top half of the program window) is written into the
“Baseline Value” control.
“Compute from graph”: If you select this option, the
baseline value is determined by averaging all points
between the “Baseline Start” and “Baseline End”
annotations. If you have not already created these
annotations, you can do so by hitting the “Cancel” button,
right-clicking over the graph where you want to start the
baseline averaging, selecting “Create Annotation” >>
“Baseline Start/End”.
STEP 4: If the “Baseline Correction” control if set to Off, turn it
On. Press the “OK” button to put your changes into effect. If you
want to save your changes to file, press the “Resave” button, select
a new filename, and then press the “OK” button.
Note 1: Whenever changes are made, the “Unsaved Changes”
button in the “Software Settings” tab is activated (turns from grey
to red). If you try to exit the program without saving your
35
changes, you will first be prompted to make sure you are aware
that your changes have not been saved.
Note 2: The Data Viewer program does not communicate with
BE3000 instruments. Changing the baseline setting from within
the Data Viewer program affects only the data read from and/or
saved to file, but does not affect the baseline settings stored in
sensor memory. If you want to change the instrument settings, the
“BE3000 Virtual Instrument” program should be used instead.
Retrospective Biomass Calibration Adjustment
The Data Viewer program also provides the capability to retrospectively
adjust the biomass calibration that is applied to the data. This feature may
be useful if the calibration settings were changed midway through a
fermentation run. During data acquisition (using the Virtual Instrument
program) such changes are applied prospectively. The Data Viewer
program allows you to apply the calibration uniformly across the entire
data file. In other situations you may wish to see the effect of applying
different calibrations to the same data set.
STEP 1: From within one of the “File” tabs, press the “Open”
button, select the file you want to work on, and then press “OK”.
STEP 2: From within the same “File” tab, press the “Adjust Cal.”
button. The “Set Calibration Retrospectively.vi” window will
open.
STEP 3: Set the “Calibration Update Method” control to one of
the four settings:
“Set Manually”: If you select this option, you should type
the new calibration settings directly into the “Calibration
Settings” control. This option is useful in circumstances in
which you have already performed a calibration fit in an
external program (e.g. Microsoft Excel).
“Retrieve from Cal. Table”: If you select this option, the
settings that are currently in the “Biomass Calibration”
table (in the top half of the program window) are written
into the “Calibration Settings” control.
“Retrieve from Cal. File”: This option is useful if you
have previously saved a calibration file, and want to apply
the same calibration settings to the currently open data file.
36
“Open Cal. Window”: Selecting this option will open the
User Calibration window. This is the same window as is
provided in the BE3000 Virtual Instrument program. See
the section “Editing, Generating, and Saving a Calibration”
for a full description of how to operate the controls in this
window.
Note: To view calibrations without having to first
open a data set, use the “Cal Window” button
provided in the “User Calibration” tab in the top
portion of the program window. Within this
window you can modify and save new calibrations
without applying these changes to a data file.
STEP 4: If the “Calibration” control if set to Off, turn it On. Press
the “OK” button to put your changes into effect. If you want to
save your changes to file, press the “Resave” button, select a new
filename, and then press the “OK” button.
Analog Output
An analog current output is available on the rear panel of the BE3000
Base Unit. This may be useful, for example, when using a third party
controller that can accept an analog output for control of the bioreactor
process. The analog output produces a standard nominal range of 4 to 20
mA and is proportional to the signal being measured by the instrument.
The analog output (AO) reflects whatever corrections are active. So if
baseline correction is on, then the AO will also be baseline-corrected.
Similarly, if user calibration is on, then the AO will be in user-calibrated
units.
A signal of zero will always correspond to a nominal value of 4 mA on the
AO. The minimum signal that corresponds to a nominal value of 20 mA
on the AO will be determined by the “analog output range” variable. This
variable can be changed from within the BE3000 Virtual Instrument
software by selecting “AO1 Range” from within the “Device
Configuration” tab near the top of the program window. Any signal
higher than this minimum signal level will cause the AO to output a
nominal value of 20 mA.
The range settings for the AO can also be set by sending serial commands
to the Base Unit. Appendices III and IV of this manual describe the serial
commands (‘R’ and ‘W’) that can be used to programmatically set this
parameter.
37
Note 1: The maximum resistance that the Base Unit can encounter to
drive a full 20 mA is 500 . By connecting a 500  resistor across the 2
analog output wires, the output can be converted from a nominal 4-20 mA
current source to a nominal 2-10 V voltage source.
Note 2: The 4 and 20 mA current levels are described above as “nominal”
values because the actual values will vary slightly from instrument-toinstrument. For best accuracy when working with the analog output, it is
recommended to measure the actual currents (or voltages) produced when
the signal is at 0 and when it is at or above the maximum determined by
the range setting. An example calibration procedure is provided in
Appendix V of this manual.
Note 3: The digital-to-analog converters (DAC) in the base unit provide
16-bit precision (meaning that the minimum step size is ~0.2 A). In
order to ensure best performance, it is important to match the Range
setting to the maximum anticipated biomass reading.
Error Codes and Averaging
Each BE3000 measurement is associated with an error code. An error
code value of zero indicates normal operation. A positive error code
indicates that a warning condition is present. When a warning condition is
present, the measurement is still expected to be reliable. A negative error
code indicates an error condition. When an error condition is present, the
measurement may be unreliable.
When working with the serial command set or the BE3000 LabView
driver, the error code is provided as the first field when a measurement
result is requested (see the ‘M’ command in Appendix IV). When
working with the BE3000 Virtual Instrument software the “Biomass
Readings” tab near the top of the window shows both the value and a
descriptive interpretation of the error code. The color of the data points on
the graph also indicates the error status (green = normal operation, yellow
= warning, red = error).
When the BE3000 instrument is on (i.e. when both the probe and USB
cable are plugged into the base unit), measurements are automatically
collected at approximately two second intervals. When the averaging time
window is set to zero, the most recent measurement is reported along with
the error code. If more than one error condition is present, the highest
priority error code is reported (see Appendix II for the prioritization of the
error codes). The ‘Ec’ command (see Appendix IV) may be used to
determine the status of all possible error conditions. When the averaging
time window is non-zero, an error is reported if more than 25% of the
38
measurements within the time window are associated with error conditions
(Note that the ‘Ne’ command can be used to change this percentage from
the default value of 25%, if desired; see Appendix IV). In this case, the
highest priority error that occurred within the time window is reported.
Warning Messages
The detectors in the BE3000 instrument are filtered so that only light near
1310 nm reaches the detector. Since light at this wavelength is strongly
attenuated by water absorbance, usually only the laser light emanating
from the tip of the probe is detected. Nevertheless, it may be possible to
overwhelm the BE3000 instrument if ambient light conditions are
extremely high. If so, you may see a “high ambient light” warning
message (error code = +1). If it is infrequent, it may be safely ignored.
If Biomass Calibration is turned on, but the present measurement is
outside of the calibration range, the “extrapolating cal” warning (error
code = +3) will be displayed. If the present reading is below the biomass
calibration range then this warning may be resolved without intervention
as the biomass increases. If the present reading is above the biomass
calibration range, you may want to consider updating the biomass
calibration with additional reference points at the high end of the biomass
range.
The effect of bubbles on the BE3000 reflectance measurements is
compensated using a two dimensional map of the central value and
distribution of the reflectance (see Principles of Operation for further
details). If the bubble conditions in your bioreactor are near the boundary
of the bubble correction map, the “below range” (error code = +4) or
“above range” (error code = +5) warning message will be displayed.
Error Messages
If the ambient light conditions are so high as to prevent accurate
measurements, the BE3000 will post an “ambient signal saturated” error
message (error code = -5). You must lower ambient lighting conditions in
order to measure accurately.
If the signal detected by the two symmetrically placed fiber optics are not
in agreement, an “interference” (error code = -12) message will be
displayed. If this error persists, make sure that the probe has at least 3 cm
of clear space in front of it. A bubble residing on the tip of the probe can
also cause this error. Bubbles will typically clear within seconds to
minutes depending on the process conditions. Placement of the probe tip
39
in high flow areas is generally recommended when bubble accumulation is
found to be a persistent problem.
If the bubble conditions in your bioreactor are outside of the boundaries of
the bubble correction map, the “bubble correction error” (error code = -13)
will be associated with the measurement. To resolve this error, it may
help to re-position the probe tip to an area of lower bubble density.
For a full listing of error codes, see Appendix II.
Maintenance
The primary maintenance task for the BE3000 instrument is cleaning of
the fiber optic interfaces. This section describes the recommended
cleaning procedures. Clearly labelled low and high reflectance standards
are provided so that proper instrument performance can be periodically
checked. Re-certification of the BE3000 instrument at BugLab is
recommended on an annual basis.
There are three fiber optic interfaces in the BE3000 instrument where
cleaning is recommended: (1) The probe tip, (2) the probe connector, and
(3) the base unit receptacle. It is important to clean the probe tip after
every usage, before it has had a chance to dry. After removal from media,
the entire immersed portion of the probe should be immediately rinsed
with distilled water, and then wiped down several times with a lint-free
tissue that has been dampened with alcohol (methanol, ethanol, or
isopropanol). Particular focus should be put on ensuring that all debris has
been removed from the tip of the probe. If in doubt, the cleanliness of the
probe can be quickly checked using the low reflectance standard (which
just contains distilled water). The raw reflectance reading on the low
reflectance standard should be less than 3, when the probe has been
properly cleaned.
A cleaning solution and two different types of cleaning sticks are available
for the purpose of cleaning the fiber optic interfaces at the probe connector
and the base unit receptacle. The cleaning sticks for the probe connector
and base unit receptacle are not interchangeable, and are intended for a
single use. Place the cleaning end of the cleaning stick over the fiber optic
cleaning solution and pump the head of the cleaning solution once to
dispense. Place the cleaning stick onto the fiber interface to be cleaned
and twist it clockwise 10 revolutions. Discard the cleaning stick and repeat
the cleaning on each fiber interface (3 fiber interfaces in the probe
connector and 3 fiber interfaces in the base unit receptacle). Cleaning of
all fiber interfaces is recommended just prior to each time the probe is
connected to the base unit receptacle. To maintain cleanliness of the
interfaces it is important to ensure that when not in use the connector and
40
receptacle are tightly covered with the protective caps, and when in use
that the two protective caps are connected to each other.
Compliance Testing
FCC
The BE3000 instrument has been tested and found to comply with
the limits of a Class A digital device, pursuant to part 15 of the
FCC Rules. These limits are designed to provide reasonable
protection against harmful interference when the instrument is
operated in a commercial environment. This equipment generates,
uses, and can radiate radio frequency energy and, if not installed
and used in accordance with the instruction manual, may cause
harmful interference to radio communications. Operation of this
equipment in a residential area is likely to cause harmful
interference in which case the user will be required to correct the
interference at his/her own expense.
ISED ICES-003 Annex
CAN ICES-3 (B)/NMB-3(B)
This Class A digital apparatus complies with Canadian ICES-003.
Cet appareil numérique de la classe A est conforme à la norme
NMB-003 du Canada.
CE Mark
This product has been assessed and found to comply against the
following standards;
EN 61326-1: 2013
--
41
End-User License Agreement
IMPORTANT—READ CAREFULLY: This End-User License
Agreement (“EULA”) is a legal agreement between you (either
individually or a single entity) and BugLab LLC (“BugLab”). By
installing, copying or otherwise using the BE3000 software, you agree to
be bound by the terms of this EULA. If you do not agree to the terms of
this EULA, Buglab is unwilling to license the BE3000 software to you. In
such an event, you may not use the BE3000 software and should contact
BugLab for instructions on the return of the product for a full refund.
Software Product License
The BE3000 software is licensed, not sold.
1. GRANT OF LICENSE. This EULA grants you the following
rights:

Software Installation and Use. You may install and use
two copies of the BE3000 software on two different
computers.

Back-up Copy. You may make one back-up copy solely
for archival purposes.
2. DESCRIPTION OF OTHER RIGHTS AND LIMITATIONS:

Limitations on Reverse Engineering, Decompilation and
Disassembly. You may not reverse engineer, decompile or
disassemble the BE3000 software.

Rental. You may not rent, lease or lend the BE3000
software.

Termination. Without prejudice to any other rights,
BugLab may terminate your rights under this EULA if you
fail to comply with the terms and conditions of this EULA.
In such an event, you must destroy all copies of the
BE3000 software.

Trademarks. This EULA does not grant you any rights in
connection with any trademarks or service marks of
BugLab or its suppliers.
42
3. COPYRIGHT. All title and intellectual property rights in and to
the BE3000 software are owned by BugLab. You may not copy
the printed materials accompanying the BE3000 software. All
rights not specifically granted under the EULA are reserved by
BugLab. Do not make illegal copies of this software.
43
BE3200 Probe Specifications
Probe (Performance)
Range of Biomass Sensitivity*
0.005 to >200 g/L
15% (biomass: 0.03-200
Biomass Accuracy*
g/L)
(typical RMSE in calibrated biomass mode)
0.005 g/L (biomass <0.03)
Averaging Time Window (trimmed mean)
2 sec - 8 min
Performance Verification/Recalibration
Reflectance standards (2)
Calibration to external reference standards
via user interface software
* Determined for dry cell weight of Saccharomyces cerevisiae during exponential
growth phase.
Probe (Environmental and Safety)
Operating Temperature (immersible
portion)
Environmental Seals (immersible portion)
Autoclaving life (with protective cap over
connector)
Laser Product Classification
4 to 100 ºC (39 to 212 F)
Water Proof
100 cycles (20 minutes at
121 ºC)
1
Probe (Physical)
Diameter (immersible portion, standard
configurations)
Length (immersible portion, standard
configurations*)
Cable Length (standard configuration*)
External Materials:
Immersible portion, shaft
Immersible portion, tip
Cable
Connector
* Custom lengths available on request
Type a: 3.2 mm (0.125”) or
Type b: 4.0 mm (0.157”) or
Type Pg13.5: 12 mm
Type a: 145 mm (5.7”) or
Type b: 205 mm (8.1”) or
Type Pg13.5: TBD
2 m (6’)
Stainless steel type 316,
N5 or better finish
Silica (core and cladding of
optical fiber), Epotek 375
epoxy
Stainless steel with silicone
over-coating
Stainless steel
Probe Accessories
High reflectance standard (contents)
High reflectance standard (shelf life)
44
SiO2 microbeads in distilled
water with 0.005%
Thimerosal (preservative)
6 months
BE3100 Base Unit Features
Base Unit (Features)
Digital (USB) input/output.
Analog (4-20mA) output with selectable range.
User Interface Software (Features)
Real-time graphical and numerical display for the sensor.
Event marking on graph, both pre-defined and user-defined.
Baseline setting and subtraction.
Factory default and user-defined biomass calibration.
Access to instrument settings.
User Interface Software (Requirements)
Windows XP SP3 / Vista / 7 / 8 / 8.1 / 10 (32 or 64 bit) Operating System.
Minimum of 256 MB of RAM.
Minimum 500 MB free hard disk space.
1024 by 768 resolution (or higher) video adapter.
Available USB communications port.
Microsoft-compatible mouse.
CD reader (required only at time of software installation).
BE3100 Base Unit Specifications
Base Unit (Electrical)
Primary DC Power In (via USB)
Optional Secondary Power In (via AC-DC
Adapter), only required if Analog Output is
used.
Certifications
Sensor Input:
Connector
Analog Output:
5 V, <150 mA
AC input to adapter: 100240 V, 50-60 Hz, <0.3 A.
DC output to base unit:
12 V, <1.0 A
CE marked. Tested for
compliance to EMC
standards EN613261:2013.
One BE3200 probe
Hybrid electro-optical: 3
multi-mode fibers and 2
electrical connections
4-20 mA (500 Ω max.)
16 bits (~0.2 A)
9 settings, logarithmically
spaced: 0.01-1,000,000
1 (2 wires)
USB
Resolution
Range Settings
Number of Outputs
Digital Output
Communications Cable:
Connectors
Length
Standard
Custom
USB (A/B)
2 m (6’)
up to 15 m (50’)
45
Base Unit (Physical)
Overall Width
Overall Length (without connectors)
Overall Height (without feet)
8 cm (3.1”)
13 cm (5.1”)
4.7 cm (1.8”)
Base Unit (Environmental and Safety)
Operating Temperature
15 to 40 ºC (59 to 104 F)
Storage Temperature
-20 to 60 ºC (-4 to 140 F)
Environmental Seals
Yes – Water resistant*
Laser Product Classification
Class 1
*When all connector ports are sealed with protective caps
46
Appendix II. Error and Warning Codes
Err.
Code
Prio
rity
Usage
(Prod
Rev)
Type
Error
Message
Associated
Variables
and
Commands
A, Ap, Ne
-17
16
3
B, G
Averaging
Error
-16
2
3
B, G
-15
3
3
B, G
-14
5
3
B, G
EEPROM
Write Error
EEPROM
Dirty Error
Reflected
Signal
Saturated
-13
15
3
B, G
Bubble
correction
error
Xx, Xz
-12
7
3
B, G
Interference
T2
-11
6
2, 3
B, G
Laser Fault
-10
9
2, 3
C
-9
10
2, 3
C
Check Sum
Error
Value out of
range
-8
11
2, 3
C
Network
busy
-7
8
2, 3
G
User
Calibration
Error
47
Description of Error
Average could not be
computed. Instead the
instantaneous results are
reported.
This error is asserted when the
laser-on signal is too high. This
should automatically be
corrected within a few
measurement cycles.
Reflectance value too high or
too low; biomass cannot be
uniquely determined or is
outside of expected range.
Detector 1 and 2 reflectance
readings do not match.
Error reported when the
maximum drive current of the
laser has been exceeded.
Sent check sum does not match
computed check sum.
This is a serial communication
error where the data packet
following a command contains
values outside of the allowed
range.
This general error code applies
when any of the 3
communicators (sensor, base
unit, or PC) is busy with
another task when any other of
the communicators is
attempting to talk with it.
Attempted to fit negative or
zero-valued data in log-log
space.
-6
17
2, 3
G
-5
4
2, 3
B, G
-4
1
2, 3
B, G
-3
13
2
B, G
User
Calibration
Error
Ambient
Signal
Saturated
Sensor
Disconnected
Below Range
-2
12
2
B, G
Above Range
-1
14
2, 3
B, G
Data Error
0
23
2, 3
B, G
1
20
2, 3
B, G
2
21
2, 3
B, G
Normal
Operation
High
Ambient
Light
No Biomass
Calibration
Insufficient number of
calibration points to compute
calibration coefficients.
Ambient light is so high that it
is preventing measurement.
Sensor not plugged into
monitor.
Signal is below the internal cal.
range
Signal is above the internal cal.
range
Error converting the individual
detector data into biomass.
Ambient light is high, but a
measurement can still be made
The biomass calibration switch
is “on”, but no biomass
calibration data is available.
3
22
2, 3
B, G Extrapolating
The measurement is outside of
Biomass Cal
the biomass calibration range.
4
18
3
B, G Below Range
Xm, Xb
Measurement is slightly below
(Bubble
the internal bubble calibration
Correction)
range.
5
19
3
B, G Above Range
Xm, Xb
Measurement is slightly above
(Bubble
the internal bubble calibration
Correction)
range.
Notes: Negative code values indicate errors; positive code values indicate warnings. In
the priority column, lower numbers indicate higher priority (1 = highest priority). In the
usage column, “2” indicates that this error code is applicable to the BE2x00 product,
while a “3” indicates that it is applicable to the BE3000 product. In the Type column, B
indicates errors that are reported by the base unit, G indicates errors reported by the
graphical user interface, and C indicates serial communication errors.
48
Appendix III.
Serial Protocol Specifications:
Remote Connection to the BE2x00
Instrument via USB or RS-232
1. Scope
Applies to communication between the BE2100 Sensor and BE2100 Base Unit,
BE2400 Base Unit, or BE|USB adapter; their communication with each other, as well
as communication protocol with a host PC.
2. General Specifications
a. Serial data is transmitted over either a RS-232 serial port or over USB.
b. Serial data is transmitted at 19200 baud rate, No Parity, 8 data bits,
1 stop bit.
c. Data transmission is non-streaming only.
d. All data will be sent as a Command-Response pair. The response key is
the lower case complement of the command key, except in the case where
the command is not recognized.
e. If a command is not recognized by the receiver then a special ASCII
character (“!”, hex: 0x21) is returned to the sender with a 1byte data
packet.
f. Empty command packets will be used to prompt for the current settings to
be returned.
g. Non-empty command packets are used to set the parameters for the
specified field value, provided the packet and parameters for the field meet
specifications. If the data in a non-empty command packet is successfully
received, the response will include a duplication of the data in the
command packet.
h. If the data in a non-empty command packet is out of the allowed range of
values, then the response will contain a 1 byte data packet with a value (9) that indicates that the data was “Out of Range”. If the computed check
sum does not match the sent check sum, then the response will contain a 1
byte data packet with a value (-10) that indicates that there was “Check
Sum Error”. If data packet is both out range and contains a check sum
error, the response data packet will indicate “Check Sum Error”. In either
case, other than responding with the error message, no action will be taken
by the receiver in response to the command sent.
i. Any 32-bit floating-point value is represented in IEEE 754 format unless
otherwise specified.
3. Data Protocol (Packet)
a. Serial data is sent in packets.
b. The packet starts with a header byte, 0x5A (ASCII character ‘Z’).
49
c. The second byte in the packet is the length byte. Length byte is the total
length of the data payload (min length of 0, max length of 127).
d. The third byte in the packet is the key byte (command and response keys
provided in Tables 1-4).
e. The next byte(s) (up to but not including the checksum) are the data bytes.
The data bytes can be zero bytes (empty packet) or up to 127 bytes of data.
The length byte, described in 4.c above, refers to the total length of these
data bytes.
f. The next-to-last byte is the checksum. The checksum chosen is a mod256
checksum byte, calculated using the key byte, data bytes and length byte.
The checksum is formed by adding the hex-value of all bytes, and
applying a modulus 256 to the sum.
g. The last byte is the footer byte, 0x3C (ASCII character ‘<’).
4. Packet Send & Receive Specifications
a. Each packet has a single message only.
b. On Power-on of the Base Unit, the Base Unit will send a single Base Unitversion packet to the serial or USB port.
c. If no sensor is connected at Base Unit power on, the Base Unit will send a
single message of “No sensor connected” to the serial or USB port.
d. On detection of a sensor, the Base Unit will send a single sensor-version
packet to the serial or USB port.
e. On detection of a sensor-disconnect status, the Base Unit will send a
single message of “sensor disconnected” to the serial or USB port.
5. Data Protocol, Message Data
a. Command keys recognized by all BE2x00 devices are shown on the left
side of Table 1. The response keys to these commands are shown on the
right side of Table 1. The commands are all ultimately received and
responded to by a BE2100 sensor. When sent to a base unit or BE|USB
adapter the commands are automatically relayed to and from the
connected BE2100 sensor(s). Further details of these commands can be
found in section 6, below.
b. Command keys recognized BE2100 Base Units, BE2400 Base Units and
and BE|USB adapters, but not by BE2100 Sensors, are shown on the left
side of Table 2. The response keys are shown on the right side of Table 2.
c. Comands recognized only by BE2100 and BE2400 Base Units are shown
in Table 3.
d. Commands recognized only by BE2400 Base Units are shown in Table 4.
e. While the Base Unit menu configuration menu is active (activated by
pressing any of the 4 keys on the pad), any commands sent to the Base
Unit will be responded to with the lower case complement of the
command plus a 1 byte data packet whose value indicates that the system
is busy (error code = -8).
f. Commands sent to the sensor while it is busy (e.g. in the middle of
running the ‘sensor check’ routine or while it is in the middle of collecting
50
baseline data) will likewise be responded to with the lower case
complement of the command sent plus a 1 byte data packet whose value
indicates that the system is busy (error code = -8).
Table 1. Commands Recognized by the BE2100 Optical Sensor Head (and passed
through by BE2100 Base Units, BE2400 Base Units, and BE|USB adapters when
connected to sensors).
Key PW
A
R/W
B
K
R/W
R/W
L
R/W
M
O
R
R/W
S
R
T
U
V
R/W
R/W
R
W
R/W
Command Description
Key
Averaging Time
Constant
Baseline Value
Start/Query/End
Baseline
‘Sensor Check’
Function Control
Get data
On/Off settings for
Base. Corr. and User
Cal.
Sensor Serial Numbers
a
b
k
l
m
o
s
User Cal Units
User Cal Coefficients
Sensor embedded SW
Version
Slope Window
t
u
v
w
!
Return Data
Description
Averaging Time
Constant
Baseline Value
Baseline status
‘Sensor Check’
Function Status
Data output
On/Off settings for
Base. Corr. and User
Cal.
Sensor Serial
Numbers
User Cal Units
User Cal Coefficients
Sensor embedded SW
Version
Slope Window
Command not
recognized
Table 2. Commands Recognized by BE2100 Base Units, BE2400 Base Units, and
BE|USB adapters.
Key PW
@
R
#
R
Command Description
Key Return Data
Description
2
Base Unit embedded
SW version
3
Base Unit Serial
Number
Get Base Unit
embedded SW version
Base Unit Serial
Number
51
Table 3. Commands Recognized only by BE2100 and BE2400 Base Units.
Key PW
J
R/W
Q
R/W
R
R/W
Command Description
Key Return Data
Description
j
Base Unit keypad
lockout state
q
Base Unit password
status
r
Range for Analog Out
Base Unit keypad
lockout state
Base Unit password
set/reset/unlock
Range for Analog Out
Table 4. Commands Recognized only by BE2400 Base Units
Key PW
%
R/W
Command Description
Sensor port switch state
Key Return Data
Description
5
Active sensor port
Notes for Tables 1-4:
(1) The symbols in the PW column indicate the type of access that is available: Read(R),
Write(W), or both (R/W).
6. Command Descriptions
a. ‘A’ – 0x41 – Averaging Time Constant
The time constant (in seconds) used in the sensor for averaging the raw detector data. The
value is represented as an unsigned integer16. Allowed values = 0, 30, 60, 120, 240, 480.
Any non-allowed value entered is ignored. Default value is 120.
Key
1 Byte
‘A’ – 0x41
Time Window
2 Bytes
Sending an empty-packet “A” command will return the “a” message, displaying the
current setting:
Key
Time Window
1 Byte
2 Bytes
‘a’ – 0x61
b. ‘B’ – 0x42 – Baseline Value
The offset value applied to the sensor ouput in order to compute the “Baseline-Corrected”
sensor output. Values expressed as 32-bit floating-point number. Default is “0.0”.
52
Key
1 Byte
‘B’ – 0x42
Baseline to Set
4 Bytes
LSB
MSB
Sending an empty-packet “B” command will return the “b” message, displaying the
current setting:
Key
Current Baseline Value
1 Byte
4 Bytes
‘b’ – 0x62
LSB
MSB
c. ‘J’ – 0x4A – Base Unit Keypad Lockout
This command is used to lock (J value = 0x01) or unlock (J value = 0x00) access to the
Base Unit keypad configuration menus. The default is unlocked. The state is always
reset to unlocked at power up of the Base Unit. When a ‘J1’ (lock base unit) command is
sent to a BE2400 (multiplexed) base unit, the base unit is automatically taken out of the
scrolling state. The scrolling state prior to sending the ‘J1’ command is saved in
memory, and automatically restored when the base unit is unlocked.
Key
1 Byte
‘J’ – 0x4A
J Value
1 Byte
Sending an empty-packet “J” command will return the “j” message, displaying the
currently set value:
Key
1 Byte
‘j’ – 0x6A
j Value
1 Byte
When communicating with BE2100 or BE2400 base units, it is recommended that the
base unit keypad be set to the “locked” state prior to performing any other
communication steps. This will prevent conflict between serial communication
commands and manually-initiated (keypad) access.
This command is only useful for BE2100 and BE2400 base units; in the case of the
BE|USB adapter no keypad access is provided, so this command serves no purpose.
d. ‘K’ – 0x4B – Start/End/Query/On-Off Baseline
This is a command from the Base Unit, BE|USB adapter, or PC to the sensor to start,
restart, end, or query the baseline status.
Key
1 Byte
‘K’ – 0x4B
K Value
1 Byte
53
K Value
0x01
0x00
0xFF
Meaning
start baseline gathering
stop baseline gathering
cancel baseline gathering
If the sensor is not gathering baseline data, setting the K value to 0x01 will command the
sensor to start gathering a baseline reading. Setting the sensor to baseline-gathering mode
will put the sensor into a mode where the averaging time constant is approximately 1 sec.
If the sensor is currently gathering baseline data, the sensor is put in a “sensor busy”
mode. This prevents the sensor from sending messages or receiving commands for the
duration of the progress, with the exception of an interrupt baseline command. An error
code is sent back with the value of “system busy”.
If the baseline is gathering data, setting the K value to 0x00 will command the sensor to
stop gathering a baseline reading. Upon completion of gathering a baseline, the baseline
(B, b) value is updated, and the Averaging Time Constant is restored to the original
value. Note that the B message is not automatically sent upon completion of calculation
of the baseline.
If the baseline is not gathering data, setting the K value to 0x00 will have no effect.
If the baseline is gathering data, setting the K value to 0xFF will command the sensor to
interrupt and cancel the gathering of a new baseline reading. Upon canceling, the
baseline (B, b) value is not updated, and the Averaging Time Constant is restored to the
original value.
If the baseline is not gathering data, setting the K value to 0xFF will have no effect.
Sending an empty-packet “K” command will return the “k” message, displaying the
currently set values:
Key
1 Byte
‘k’ – 0x6B
K Value
1 Byte
The returned values are either 0x01 (currently collecting baseline data) or 0x00 (not
collecting baseline data).
e. ‘L’ – 0x4C – ’Sensor Check’ Function Control
This is a command from the Base Unit, BE|USB adapter, or PC to the sensor to start, end,
or apply the results of the ‘Sensor Check’ function. The Sensor Check function runs
from within the sensor embedded software. The purpose of the ‘Sensor Check’ routine is
to determine if the sensor is performing correctly, and, if necessary, reset the internal
54
sensor calibration. A one-byte value following the ‘L’ command provides the control for
the ’Sensor Check’ routine.
Key
1 Byte
‘L’ – 0x4C
L Values
0x00
0x01
0x02
0x03
0xFF
L Value
1 Byte
Meaning
Clear data.
Initiate Low cal cup measurement.
Initiate High cal cup measurement.
Use cal cup measurements to set new Variable Sensor Coefficients.
Interrupt measurement and resume normal mode.
Note: values not defined above are illegal and ignored by the sensor.
Sending an empty ‘L’ command packet will return the ‘l’ message along with a data
packet. The first Byte of the packet holds a status message. The meaning of the status
messages are shown below:
l Values
0x00
0x11
0x12
0x13
0x21
0x22
0x31
0x32
0x41
0x42
0x53
0x63
Meaning
No data.
Busy with Low cal cup measurement.
Busy with High cal cup measurement.
Busy with setting new Variable Sensor Coefficients.
Low cal cup measurement was unstable.
High cal cup measurement was unstable.
Low cal cup measurement was stable but Combined Result failed.
High cal cup measurement was stable but Combined Result failed.
Low cal cup measurement was stable and Combined Result passed.
High cal cup measurement was stable and Combined Result passed.
Error - new Variable Sensor Coefficients could not be set.
New Variable Sensor Coefficients were successfully set.
If the ‘Sensor Check’ routine has just been started (either through the interactive keypad
or the user interface software) and no calibration cup measurements have been initiated
yet, sending an empty ‘L’ command to the sensor will result in an “l value” of 0x00 (No
data) being returned.
If a calibration cup measurement is in progress when an empty ‘L’ command is sent to
the sensor, a ‘Busy’ (0x11 or 0x12) “l value” will be returned to the sender. When a
calibration cup measurement has been completed, sending an empty ‘L’ command to the
sensor will result in one of 6 “l values” being returned. The returned “l value” can be
used to determine the type of calibration cup that was measured (“low” or “high”),
whether the standard deviation of the 10 calibration cup measurements was above
(“unstable”) or equal to or below (“stable”) a threshold value. In the case that the
55
measurement was stable, the “l value” further indicates whether or not the sensor result,
when compared to the result computed from the stored Calibration Cup Coefficients, had
an absolute error that exceeded (“failed”) or was equal to or below (“pass”) a threshold
value.
If calculation and saving of new Variable Sensor Coefficients is in progress when an
empty ‘L’ command is sent to the sensor, a ‘Busy’ (0x13) “L value” will be returned to
the sender. When new Variable Sensor Coefficients have been computed and saved,
sending an empty ‘L’ command to the sensor will result in an “l value” of 0x63 being
returned. When an error is encountered during the calculation or saving of new Variable
Sensor Coefficients, sending an empty ‘L’ command to the sensor will result in an “l
value” of 0x53 being returned.
In addition to the 1 Byte ‘l value’, 2 32-bit floats are returned in response to an empty
packet “L” command. The first float value is the sensor response (in “Bug Units”)
measured on the calibration cup; the second float value is the stored sensor response.
The “set new variable sensor coefficients” command should only be sent when “stable”
measurements have been completed on both the Low and High Calibration Cups. When
the cal cup measurement is interrupted (by sending an L value of 0xFF) the calibration
cup measurement data will be cleared from sensor memory with no change to the
Variable Sensor Coefficients. Sending an empty packet “L” query to the sensor after
sending and “L” command with an “L Value” of either the 0x00 (clear data) or 0xFF
(interrupt and resume normal mode), will result in an “l value” of 0x00 (no data) being
returned.
Returned ‘l’ packet in ‘normal’ mode
Key
l Value
Measured Combined
Stored
Result
Combined Result
1 Byte
1 Byte
4 Bytes
4 Bytes
‘l’ – 0x6C
MSB
LSB MSB
LSB
When the ‘Sensor Check’ routine is running in the sensor, all commands other than an
‘L’ command will be responded to with a “system busy” 1 byte error code.
f. ‘M’ – 0x4D – Data Request
This command is used to request a data packet from either the sensor or the Base Unit.
Returns the sensor data output. The only format for the ‘M’ command is an empty ‘M’
command.
Key
1 Byte
‘M’ – 0x4D
Sending an empty-packet “M” command will return the “m” message, along with a data
packet. The data packet has 6 components:
56
(1)
(2)
(3)
(4)
(5)
(6)
Error Code (8-bit signed integer),
Raw Sensor Measurement Result (in ‘Bug Units’) (32-bit floating point),
Baseline-Corrected Measurement Result (32-bit floating point),
User Calibrated Result (32-bit floating point),
Growth Rate Constant (1/hours) (32-bit floating point),
User Calibration Units (up to 11 ASCII bytes).
Key
Error
Code
Raw Result
Base.-Corr.
Result
User Cal
Result
Growth Rate
(1/hrs)
1 Byte
1
Byte
4 Bytes
4 Bytes
4 Bytes
4 Bytes
‘m’ –
0x6D
LSB
MSB
LSB
MSB
LSB
MSB
LSB
MSB
Note: the total length of the data packet is variable due to the variable length of the ‘User
Cal Units’ string. The ‘User Cal Units’ string includes a terminating null character as its
last byte. Therefore, the maximum allowed number of non-null characters in the string is
10.
g. ‘O’ – 0x4F – On/Off Setting for Baseline Corr. and User Cal.
The ‘O’ command is used to turn On or Off Baseline Correction and User Calibration.
Values are represented as 8-bit unsigned integers. Allowed values are 0 (Off) and 1 (On),
with the default value being 0 (Off) for both functions. The first byte sets the On/Off
state for Baseline Correction. The second byte sets the On/Off state for User Calibration.
Sending the highest value for a byte (hex ‘FF’) will preserve the current setting in the
byte. Values other than 0, 1, and F will be ignored.
Key
1 Byte
‘O’ – 0x4F
Baseline Correction
On/Off
1 Byte
User Calibration
On/Off
1 Byte
Sending an empty-packet “O” command will return the “o” message, displaying the
currently set values:
Key
Baseline Correction
User Calibration
On/Off
On/Off
1 Byte
1 Byte
1 Byte
‘o’ – 0x6F
57
User
Cal.
Units
up to11
ASCII
Bytes
…
h. ‘Q’ – 0x51 – Base Unit Password Settings
You may wish to set up password-protected access to the keypad functions on the Base
Unit. When in the locked state, entry to the Base Unit functions is enabled by entering a
six-number value via the keypad. Only values from 1 to 4 are valid for each number. If
the six numbers (ASCII) entered on the keypad match the currently-stored password, the
system enters an unlocked state until the user exits the configuration menu. If the
numbers entered do not match the currently-stored password, access to the configuration
menus is denied, and returns to the normal display. The value of the Base Unit password
is stored in the Base Unit. The default password is ‘111111’. In addition to changing the
password via the keypad on the Base Unit, the password may be set through the ‘Q’
command:
Key
1 Byte
‘Q’ – 0x51
P-Value1 P-Value2 P-Value3 P-Value4 P-Value5 P-Value6
1 Byte
1 Byte
1 Byte
1 Byte
1 Byte
1 Byte
The Q-command may also be used to reset and turn On or Off password protection by
sending a 1-byte value for a Q-command. Sending a 0x00 turns password protection Off.
Sending a 0x01 turns password protection On. Sending a 0x02 resets the password to
“1,1,1,1,1,1” and turns Off password protection. Sending a 0x03 resets the password to
“1,1,1,1,1,1” and turns On password protection. Note that the values for “1,1,1,1,1,1” are
represented in hex as a six-element array of 0x31.
Key
1 Byte
‘Q’ – 0x51
QCommand
1 Byte
Sending an empty-packet “Q” command will return the “q” message, displaying the
currently set status:
Key
1 Byte
‘q’ – 0x71
q-Value
1 Byte
Valid return values are locked (0x00) or unlocked (0x01). This command is only useful
for BE2100 and BE2400 base units; in the case of the BE|USB adapter no keypad access
is provided, so this command serves no purpose.
i. ‘R’ – 0x52 – Range for Analog Output
Sets the range for the analog output terminals of BE2100 and BE2400 Base Units.
Values are represented as 8-bit unsigned integers. Allowed values are 0-5 with a default
value of ‘4’. The table below shows the correspondence between the integer
representation and the range value:
Integer Representation
Range Value
58
0
1
2
3
4
5
0.01
0.1
1.0
10.0
100.0 (default)
1000.0
The first byte is the range for Analog Out 1. The second byte is the range for Analog Out
2 (BE2100 Base Units only). Sending the high value (hex FF) for a byte will preserve the
current setting in that byte. Byte values other than 0, 1, 2, 3, 4, 5 and FF are ignored.
Key
1 Byte
‘R’ – 0x52
AO1&2 Range
1 Byte
1 Byte
AO1
AO2
Sending an empty-packet “R” command will return the “r” message, displaying the
currently set values:
Key
1 Byte
‘r’ – 0x72
AO1&2 Range
1 Byte
1 Byte
AO1
AO2
The above description applies only to the “standard” configuration where a sensor is
connected to a BE2100 base unit. If a BE2400 (multiplexing) base unit is used instead,
only one analog output is available for each sensor (AO1); changing the range setting for
AO2 has no effect. If the sensor is connected via a BE|USB adapter no analog output is
available, so the range command serves no purpose.
j. ‘S’ – 0x53 –Sensor Serial Number
This command reads the Sensor Serial Number. The field data is represented as a 32-bit
unsigned integer. Sending an empty-packet ‘S’ command will return the ‘s’ message,
displaying the currently set values. It is also sent automatically when the device is first
powered up (following the send of the Base Unit or BE|USB embedded software version
data).
Key
1 Byte
‘s’ – 0x73
Sensor S/N
4 Bytes
MSB
LSB
k. ‘T’ – 0x54 – User Calibration Units
Null-terminated string of up to 11 characters describing the user-calibration units (e.g.
‘mg/L e coli’). Default setting is “Cal Units”. Non-printable ASCII characters are
ignored.
Key
User Cal Units
59
1 Byte
‘T’ – 0x54
(up to) 11 ASCII Bytes
…
Sending an empty-packet “T” command will return the “t” message, displaying the
current string:
Key
1 Byte
‘t’ – 0x74
User Cal Units
(up to) 11 ASCII Bytes
…
The last byte is to be used for the null-termination character, so the maximum useful
string length is 10 characters. Note: The response string contains 1 more character than
the command string; this last response character should be ignored.
l. ‘U’ – 0x55 –User Calibration Coefficients
Six 32-bit floating-point values and one unsigned integer: offset, linear coeff, quadratic
coeff, cubic coeff, MinX, and MaxX, and Transform Method. Min and Max X are the
minimum and maximum values of the Sensor Measurement Results used in generating
the calibration coefficient. These values are useful for determining whether the
calibration is being extrapolated beyond the range of the calibration data. Transform
Method is either linear (value = 0) or log (value = 1). Defaults are defined as follows:
[0.00, 1.00, 0.00, 0.00, 0.00, 0.00]
Key
1 Byte
‘U’–
0x55
Offset
4 B yt e s
LSB
Linear Coeff
4 B yt e s
MSB LSB
MSB
Quadratic Coeff
Cubic Coeff
4 B yt e s
4 B yt e s
LSB
MSB LSB
MSB
MinX
4 Bytes
LSB
MaxX
4 Bytes
MSB
LSB
Transform Method
1 Byte
MSB
(the coefficients appear as a continuous message; this table has been formatted to fit on
the page).
Sending an empty-packet “U” command will return the “u” message, displaying the
currently set array:
Key
Offset
Linear Coeff
1 Byte
4 B yt e s
4 B yt e s
‘u’– 0x75 LSB
MSB LSB
MSB
60
Quadratic Coeff
Cubic Coeff
4 B yt e s
4 B yt e s
LSB
MSB LSB
MSB
MinX
4 B yt e s
LSB
MaxX
4 B yt e s
MSB LSB
MSB
(the coefficients appear as a continuous message; this table has been formatted to fit on
the page).
m. ‘V’ – 0x56 – Sensor Embedded Software Version
This command reads the version of the embedded software that exists in the sensor. The
data field is comprised of a 32 bit floating point value. The only format for the ‘V’
command is an empty ‘V’ command.
Key
1 Byte
‘V’ – 0x56
Sending an empty-packet ‘V’ command will return the ‘v’ message, displaying the
current setting:
Key
1 Byte
‘v’ – 0x76
Sensor S/W version
32 bits
LSB
MSB
n. ‘W’ – 0x57 – Growth Rate Window
The ‘W’ command determines the time window (in seconds) to be used when estimating
the exponential growth rate contant. The data field is comprised of an unsigned integer16.
Any value that is below the Averaging Time Constant (see the ‘A’ command) is set to the
Averaging Time Constant for windowing. Allowed values are: 60, 120, 240, 480, 960,
and 1920. The default value is 480.
Key
1 Byte
‘W’ – 0x57
Slope Window
2 Bytes
MSB
LSB
Sending an empty-packet “W” command will return the “w” message, displaying the
current setting:
61
Key
1 Byte
‘w’ – 0x77
Slope Window
2 Bytes
MSB
LSB
o. ‘@’ – 0x40 – Base Unit Embedded Software Version
This command reads the version of the embedded software that exists in the Base Unit.
This data is sent automatically when the device is first powered up. The data field is
comprised of a 32 bit floating point value. The only format for the ‘@’ command is an
empty ‘@’ command.
Key
1 Byte
‘@’ – 0x40
Sending an empty-packet ‘@’ command will return the ‘2’ message, displaying the
current setting:
Key
1 Byte
‘2’ – 0x32
Base Unit S/W version
32 bits
LSB
MSB
p. ‘#’ – 0x23 –Base Unit Serial Number
This command reads the Base Unit Serial Number. The field data is represented as a 32bit unsigned integer. Sending an empty-packet ‘#’ command will return the ‘3’ message,
displaying the currently set values.
Key
1 Byte
‘3’ – 0x33
Base Unit S/N
4 Bytes
MSB
LSB
62
Appendix IV.
Serial Protocol Specifications:
BE3000 Deviations from BE2x00 Serial
Protocol
1. Scope
Except as noted in this document, the BE2x00 serial protocol is also applicable to the
BE3000 product.
2. Overview
All amendments to the BE2x00 serial protocol for the BE3000 product series are
made with the goal of minimizing changes to the BE2x00 protocol. All BE2x00
commands are a single letter. The BE3000 command set is extended by use of a meta
command (‘Z’) that is used for BE3000 devices only, and provides access to an
extended 2-letter command set.
Table 1. Summary of BE3000 Communication Commands
Name Function
PW3 M4 A5 Type
A
Window size (in
R/W
P
S UInt16
seconds) used in the
trimmed mean filter.
Ac
Erases the history
R/W
S buffer for the “A”
command
Ao
Sets the analog
R/W
P
S UInt8
output state: 0 = low,
1 = high, 2 = linearly
proportional, 3 =
log10 proportional
B
Baseline value
R/W
P
S Flt32
Ch
Selected channel that R/W B
G UInt8
all subsequent serial
commands will
interact with.
Cr
Scanned channel
R/W B
G UInt8,
range. The first
UInt8
argument is the
starting channel.
The second
argument is the last
channel +1.
Cx
Factory default
R
B
G UInt8, 1biomass calibration
32 bytes
63
Default
120
Units
seconds
-
-
2
-
0.0
0
Depends
on product
version
(see full
descript.)
“Unused”
Channel
index
(zero
based)
Channel
index
(zero
based)
0-9,
ASCII
Dx
Ec
J
K
L
M
Ne
NO
O
Q
R
S
T
units. This command
has the same format
as the “T” command,
except the
calibration number
(0-9) is first
referenced.
User-defined
biomass calibration
units. This command
has the same format
as the “T” command,
except the
calibration number
(0-9) is first
referenced.
Status settings for all
error bits.
Not currently used.
Start/query/end
baseline.
‘Sensor Check’
function control.
Get data command.
See command
description below.
Max percentage of
allowed errors
The number of
outliers in a row that
results in error being
reported.
On/Off settings for
baseline correction
and user calibration.
Not currently used.
Analog output range.
Note: only the 1st
field is currently
used.
Probe serial number.
The currently active
user calibration
units.
characters
, nullterminate
d
R/W
B
G
UInt8,
1-32 bytes
“Unused”
0-9,
ASCII
characters
, nullterminate
d
R
B
S
UInt32
0 when no
errors
Error bits
R/W
S
1 byte
R/W
S
R
S
%
R/W
B
G
UInt15
25
R/W
B
G
UInt16
1
R/W
P
S
2x
Byte
[0, 0]
R/W
B
S
2 x UInt8
[1000.0,
100.0]
R
P
S
R/W
P
S
UInt32 or
UInt64
1-32 bytes
32000000
0
“Unused”
64
Depends
on ‘O’
settings
ASCII
characters
, null-
terminate
d
U
The currently active
biomass calibration
coefficients.
R/W
P
S
6 x Flt32,
UInt8
Un
Select user
calibration set. The
two fields are Bank
(0=factory, 1=user),
and Calibration
Selection (0-9).
Factory default
biomass calibration
coefficients. This
command has the
same format as the
“U” command,
except the
calibration number
(0-9) is first
referenced.
Firmware version.
See the command
description below.
User-defined
biomass calibration
coefficients. This
command has the
same format as the
“U” command,
except the
calibration number
(0-9) is first
referenced.
Slope window.
Meta command
providing access to
the extended (2letter) command set.
See below.
Base unit serial
number. Format
3100xxxxx where
xxxxx starts at
00001 and
R or
R/W
B
S
2 x UInt8
R
B
G
UInt8, 6 x
Flt32,
UInt8
[0, 0.0,
1.0, 0.0,
0.0, 0.0,
0.0,
0]
R
B
G
R/W
B
G
UInt8, 6 x
Flt32,
UInt8
[0, 0.0,
1.0, 0.0,
0.0, 0.0,
0.0,
0]
R/W
P
S
Int16
480
R
B
S
UInt32
31000000
0
Ux
V
Vx
W
Z
#
65
[0.0, 1.0,
0.0, 0.0,
0.0, 0.0,
0]
[0, 0]
[0-1, 0-9]
seconds
increments in steps
of 1.
Notes for Table 1:
(1) All 1-letter commands are also BE2x00 commands. See the BE2x00 serial
protocol for further information. Deviations from BE2x00 usage are described
below.
(2) All 2-letter commands are unique to the BE3x00 product. See below for further
information.
(3) The symbols in the PW column indicate the type of access that is available:
Read(R), Write(W), or both (R/W).
(4) The symbols in the M column indicate where the data associated with this
command is to be stored: Probe (P), Base Unit (B), or Hard Coded (H).
(5) The symbols in the Applicability (A) column indicate whether the command is
specific (S) to the currently active sensor, or applies globally (G) across the
instrument. For those commands that are specific (S), the active sensor must first
be selected by using the “Ch” command.
3. Data Protocol (Packet) for Extended Command Set
a. The data protocol for the extended command set is the same as the 1 byte
command set, except as follows.
i. The third byte is the ‘Z’ command.
ii. The next 2 bytes are the extended command bytes (big-endian
uint16 - 2 bytes).
iii. The next bytes are the optional data bytes for the extended
command.
iv. The maximum length of the data payload is 255 bytes (127 bytes
for BE2x00 protocol).
v. The extended command is considered to be part of the “data
packet”, so the length byte will include the 2 bytes of the extended
command (in both the command and response).
vi. The first letter of the response will be “z” followed by an echo of
the extended command (no case change from the extended
command).
b. Example command byte stream: 'Z',0x02,'Z',0x00,0x01,0xE4,'<'
This is extended command #0x0001 with no optional arguments.
7. Command Descriptions
All single letter commands follow the same protocol as specified in Reference
2.a.v, except as specified below. All 2-letter commands are unique to the BE3000
product series.
a. ‘A’ – Measurement Averaging Time Window
Unlike the BE2x00 product which uses an IIR filter, the “average” computed by the
BE3000 uses a trimmed mean filter. The averaging is applied to the bubble-corrected
66
reflectance measurements (R0) before baseline correction or biomass calibration is
applied. As measurements are being built up in the history buffer, the number of samples
that are currently available are used to compute a trimmed mean, until the history buffer
is full. The history buffer is cleared after events that are expected to have an
instantaneous effect on the signal: laser power and detector gain adjustment. The
history buffer can also be cleared by sending the “Ac” command. For sequentiallymultiplexed sensors, such as sensors attached to a BEPM3000 base unit, the number of
measurements contributing to the average will vary according to the number of sensors
being multiplexed-between. In contrast, for BE2100 sensors attached to a BE2400 base
units, which is parallel multiplexed, the number of samples contributing to each
measurement will be independent of the number of attached sensors. The range of
allowed time windows and the default value is the same as for the BE2x00 product.
b. ‘Ac’ – Clear Averaging History
Erases the averaging history buffer for the selected channel. This command has no
arguments.
a. ‘Ao’ – Analog Output
Set/gets the analog output state. Allowed states:
0 – Analog output is set to the lowest current (nominally 4 mA).
1 – Analog output is set to the highest current (nominally 20 mA).
2 – Analog output is linearly proportional to the sensor signal (this is the default value).
3 – Analog output is proportional to log10 of the sensor signal.
The sensor signal to which the analog output is proportional depends on the ‘O’ settings.
The argument type is UInt8. This setting is local to each sensor, and is stored in sensor
memory. The 0 and 1 settings do not persist across power cycling; instead the setting is
returned to being proportional, either linear or log10, whichever was most recently active
for that sensor channel.
When ‘Ao’ is set to 3 (log10 output), the maximum range (‘R’ command) is still defined
in linear units (i.e. prior to taking the log10), and the minimum value (corresponding to 4
mA current and also defined in linear units) is defined as 1x10-6 times the range setting.
b. ‘Ch’ – Selected Channel
For multiplexed versions of the BE3000 product, the “Ch” command selects the active
channel that all subsequent serial commands will interact with. Error code 1 is returned
the selected sensor is out of range with respect to the selected product (i.e. single channel
product will only accept channel “0”). The data packet is a single byte (UInt8). The
default value is 0.
c. ‘Cr’ – Scanned Channel Range
For multiplexed versions of the BE3000 product, channels are continuously scanned (1
measurement on each channel) by the firmware. The “Cr” command determines the
range of channels that are scanned. The data packet consists of 2 UInt8: the start
channel, and the end channel plus one. The channel index is zero based. If both
arguments are the same, then no channels are scanned. For example, to scan the first 4
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channels, use: 0, 4. The default values for this command depends on the product
version:
Product Version
Default Start Channel
Default End Channel
ProBE single
0
1
ProBE mux
0
8
a. ‘Cx’ – Factory Default Biomass Calibration Units
Up to 10 factory default biomass calibrations are stored in the memory of the BE3000
base unit. This command provides access to the factory default biomass calibration units
and has the same format as the “T” command, except the calibration number (UInt8,
allowed values: 0-9) is first referenced. Null-terminated string fields up to 32 characters
long are supported. The last byte is to be used for the null-termination character, so the
maximum useful string length is 31 characters. The default value is “Unused”. The
command is read only.
b. ‘Dx’ – User Defined Biomass Calibration Units
Up to 10 user-defined biomass calibrations are stored in the memory of the BE3000 base
unit. This command has the same format as the “T” command, except the calibration
number (UInt8, allowed values: 0-9) is first referenced. Null-terminated string fields up
to 32 characters long are supported. The last byte is to be used for the null-termination
character, so the maximum useful string length is 31 characters. The default value is
“Unused”.
c. ‘Ec’ – Status setting of all error and warning codes
The bit state of all errors and warnings encoded as a logical OR of all error and warning
codes, in 32-bit un-signed integer format in big endian byte order. The table below
shows the bit mapping:
Bit
Hex value
Error
Detector
Description
number
Code
1
0x00000001
-13
1
D1 unstable (bubble corr.)
2
0x00000002
-12
Optical interference
3
0x00000004
-11
Laser fault
4
0x00000008
-8
Busy (comm.)
5
0x00000010
-5
1
D1 ambient light saturated
6
0x00000020
-3
Below range (bug units cal.)
7
0x00000040
-2
Above range (bug unit cal.)
8
0x00000080
-1
Math (bug units cal.)
9
0x00000100
1
1
D1 high ambient light
10
0x00000200
3
Extrapolating cal. (biomass cal.)
11
0x00000400
4
1
D1 below range (bubble corr.)
12
0x00000800
5
1
D1 above range (bubble corr.)
13
0x00001000
-4
Sensor disconnected
14
0x00002000
-14
1
D1 reflectance saturated
15
0x00004000
-13
2
D2 unstable (bubble corr.)
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16
17
18
19
20
21
22
23
24
0x00008000
0x00010000
0x00020000
0x00040000
0x00080000
0x00100000
0x00200000
0x00400000
0x00800000
4
5
-5
1
-14
-7
-17
-16
-15
2
2
2
2
2
-
D2 below range (bubble corr.)
D2 above range (bubble corr.)
D2 ambient light saturated
D2 high ambient light
D2 reflectance signal saturated
Math (biomass cal.)
Averaging failed
EEPROM write error
EEPROM dirty error
a. ‘K’ – Baseline Collection
Removed the BE2x00 requirement that a low calibration cup measurement must preceed
a high calibration cup measurement. Removed the BEx200 requirement that all other
commands are locked out while sensor check is busy. For the BE3000, only the ‘L’
command is locked out for a particular sensor while Baseline Collection (‘K’ command)
is in progress on that sensor. The ‘K’ command will return “busy” while Sensor Check
measurements (‘L’ command) are actively being collected on the same sensor. In that
event, the data packet in the response to the ‘K’ command will consist of a single byte
having a value of xF8.
a. ‘L’ – Sensor Check
Removed the BE2x00 requirement that a low calibration cup measurement must preceed
a high calibration cup measurement. Removed the BEx200 requirement that all other
commands are locked out while sensor check is busy. For the BE3000, only the ‘K’
command is locked out for a particular sensor while a low or high reflectance standard
Sensor Check (‘L’ command) measurement is in progress on that sensor. The ‘L’
command will return “busy” while baseline collection (‘K’ command) is active on the
same sensor. In that event, the data packet in the response to the ‘L’ command will
consist of a single byte having a value of xF8.
b. ‘M’ – Data Request
The “Combined Result” that is reported to the user is the bubble-corrected reflectance,
R0. The biomass calibration units field is no longer included in the ‘M’ response. The
error code is an intelligent summary of the error bits due to averaging. If “Ne” percent or
less of the measurements in the history buffer encounter an error, then no error will be
reported. If more than “Ne” percent of the measurements encounter an error, then the
highest priority error code of any error bit seen in recent history is reported. For example,
if the full history buffer contains 16 samples and the Sensor error bit is asserted on one
measurement in the history, and the Interference error bit is set on 3 other measurements:
- If Ne=25%, no error will be reported
- If Ne = <25%, error code Sensor (-4) will be asserted
If the averaging parameters (Ap for example) are out of range, then
AVERAGING_ERROR will be reported, and the last instantaneous value reported.
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c. ‘Ne’ – Maximum Percentage of Allowed Errors
‘Ne’ determines the maximum percentage of errors in recent history (with the history
window defined by the ‘A’ command) that are allowed to not be reported by the ‘M’
command. If ‘Ne’ percent or less measurements in the history buffer encounter an error,
then no error will be reported by the ‘M’ command. If more than ‘Ne’ percent of the
measurements encounter an error, then the highest priority error code of an error bit seen
in the recent history will be reported. No averaging error is reported when the averaging
time window (‘A’) is set to zero.
d. ‘NO’ – Threshold Number of Sequential Outliers
When detectors 1 and 2 are not in agreement (such as due to the presence of a nearby
reflective object), the measurement is flagged as outlier. The “NO” command determines
the number of sequential outliers that must be detected before the outlier error bit is set
(error code: -12). The default value is 1.
e. ‘R’ – Analog Output Range
The BE3000 provides an analog output that is proportional to the sensor signal, but not a
growth rate output, so only the first range setting is used. The default setting for the
analog output range is 5 (1000.0). Three additional high range settings (beyond those
provided in the BE2x00 product line) are available in the BE3000 product, as shown
below:
Integer Representation
0
1
2
3
4
5
6
7
8
Range Value
0.01
0.1
1.0
10.0
100.0
1000.0 (default)
10,000.0
100,000.0
1,000,000.0
When ‘Ao’ is set to 3 (log10 output), the maximum range (‘R’ command) is still defined
in linear units (i.e. prior to taking the log10), and the minimum value (corresponding to 4
mA current and also defined in linear units) is defined as 1x10-6 times the range setting.
f. ‘T’ – Biomass Calibration Units
This variable is now just a pointer to the Active biomass calibration units (“Cx” or “Dx”),
as determined by the “Un” command. Null-terminated string lengths of up to 32
characters are now supported. The last byte is to be used for the null-termination
character, so the maximum useful string length is 31 characters. The default setting is to
point to the first string of the default biomass calibration units (Cx[0]). This command is
read-only.
70
g. ‘U’ – Active Biomass Calibration Coefficients
The ‘U’ command does not read its own coefficients. It reads the coefficients pointed at
by the new ‘Un’ command. By default the ‘U’ command points to the first set of factory
default biomass calibration coefficients (Ux[0]). This command is read-only.
h. ‘Un’ – Select Biomass Calibration Set
Up to 20 sets of biomass calibration coefficients are stored by the Ux (Factory Default)
and the Vx (User Defined) commands. The “Un” command specifies which calibration is
currently active, using two UInt8 fields: (1) Bank, and (2) Selection. The Bank field
specifies whether the active calibration is in the Factory Default (Bank = 0) or the UserDefined (Bank = 1) set. The Selection field (= 0-9) specifies which of the 10 fields
within the Bank is active. In addition to selecting the active biomass calibration
coefficients, the active biomass units (“Cx” and “Dx”) are also selected.
i. ‘Ux’ – Factory Default Biomass Calibration Coefficients
Up to 10 factory default biomass calibrations are stored in the memory of the BE3000
base unit. This command provides access to the factory default biomass calibration
coefficients and has the same format as the “U” command, except the calibration number
(UInt8, allowed values: 0-9) is first referenced. This command is read-only.
j. ‘V’ – Embedded Software Version
The BE2x00 serial protocol provides separate commands for accessing the sensor (“V”)
and base unit (“#”) firmware versions. In the BE3000 product, the ProBE does not
contain a microprocessor or firmware, so only one command (“V”) is needed for
retrieving the firmware version. In the BE2x00 product the version number is stored as a
single precision (32 bit) float value. In the BE3000 product the version number is stored
as an unsigned 32 bit integer, and can be converted to a float by dividing by 100.
k. ‘Vx’ – User-Defined Biomass Calibration Coefficients
Up to 10 factory default biomass calibrations are stored in the memory of the BE3000
base unit. This command provides access to the user-defined biomass calibration
coefficients and has the same format as the “U” command, except the calibration number
(UInt8, allowed values: 0-9) is first referenced.
l. ‘Z’ – Extended Command Set
Key
Extended
Command Set
1 Byte
‘Z’ – 0x5A
[Additional data,
specific to the
particular extended
command]
2 Bytes
MSB
LSB
Sending an empty-packet ‘Z’ command shall not be recognized. Sending a ‘Z’ command
and one of the 2-letter extended commands with no additional data shall return the ‘z’
71
message, followed by the extended command and the current setting for the extended
command.
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Appendix V. Example Procedure for Calibrating the Analog
Output
The analog output (AO) on the BE3000 base units provides a current output that is
proportional to biomass. The nominal range of the current is 4 to 20 mA. However, due
to component variation, the actual minimum and maximum currents vary somewhat
between base units. Therefore, when using the AO outputs, it recommended that the
actual minimum and maximum currents be measured and used as calibration inputs for
the AO reading device (such as an analog-to-digital converter on your bioreactor
controller). The following example provides a step-by-step method for measuring the
minimum and maximum AO currents.
Tools needed:
- DC Ammeter (with measurement range of ~1 mA to at least 20 mA).
- Analog output cable with lemo connector on one end and bare wire on the other end.
- low and high reflectance standards (as provided with BE3200 probes).
Procedure:
1. Set the averaging time constant on the base unit to zero.
 In the BE3000 software, under the Ave. Time column within the Device
Configuration tab, select “0”.
2. Set the Range for the analog output you wish to calibrate to 100.
 In the BE3000 software, under the AO1 Range column within the Device
Configuration tab, select “100”.
3. Turn Off both Baseline Correction and User Calibration
 In the BE3000 software, under both the Base Corr and the Biomass Cal columns,
select “Off”.
4. Remove the protective cap from the tip of the probe. Place probe tip into the low
reflectance standard. Insert the probe just below the neck of the tube and make sure
that no bubbles are on or near the tip of the probe.
5. Attach one of the bare wire leads of the analog output cable into the device into which
it is to be read (such as an analog-to-digital converter on your bioreactor controller).
6. Attach the ammeter between the 2nd bare wire lead of the analog output cable and the
2nd input connection of the device into which it is to be read.
7. Using the Ammeter, measure the current. Record this as A(low).
8. Thoroughly mix the contents of the high reflectance standard by shaking and/or
vortexing until no sediment can be seen on the sides or bottom of the tube, and then
shake/mix for several additional seconds. Remove the probe from the low reflectance
73
standard, wipe the tip with a lint-free tissue, and then insert it into the high reflectance
standard in the same manner as it was inserted into the low reflectance standard.
9. Using the Ammeter, measure the current. Record this as A(high).
10. Return the sensor averaging time constant, range, baseline correction, and biomass
calibration settings to their prior settings.
11. Use I(low) and I(high) as calibration inputs for your analog reading device (e.g.
bioreactor controller). This step will vary depending on the control software you are
using, but typical linear calibration inputs are “offset” and “span” values in units of
current and corresponding biomass units. The table below shows the correspondence
between offset and span and the values you just measured.
Table 1. Example Linear Analog Calibration Inputs
Current (mA)
I(low)
Offset
I(high)-I(low)
Span
Biomass
0.00
BE3000 base unit Range
setting
Note that the Range value you use for the Biomass Span should be the Range setting
that you will select during your bioreactor run (not the Range setting just used during
the calibration procedure). If you change the range setting on the BE3000 base unit,
you will also need to update the calibration in your control software. Also, the table
above assumes that baseline correction will be applied to the BE3000 result so that
when media alone is measured, the biomass reading is zero. If baseline correction is
not applied and the biomass reading for media alone is not zero, you should use this
baseline reading as the Biomass Offset and subtract this value from the span.
However, baseline-correction via the BE3000 instrument is generally recommended,
since this can be applied without having to update the calibration inputs for your
control software.
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Appendix VI. Trouble-Shooting
Probe Trouble-Shooting
Observation
Disagreement between
BE3000 instrument and
offline reference
measurement
Possible Causes
(1) Incorrect biomass
calibration selected.
(2) Incorrect baseline.
(3) Probe tip is dirty.
(4) Cell lysates are
contributing significantly to
the measured optical
reflectance.
Sensor Check test failed.
(1) Low reflectance solution
is not clean.
(2) Probe tip is not clean.
(3) Laser aging.
Biomass readings are not
stable.
(1) Probe position needs to be
optimized.
(2) The sensor averaging time
constant needs to be
optimized.
Probe Calibration Trouble-Shooting
Observation
Possible Causes
“Extrapolating Cal”
The displayed biomass is
outside the range of the usergenerated calibration.
75
Suggested Remedies
(1) See User Calibration.
(2) Collect a baseline
reading on the media alone,
prior to inoculation. See
Setting the Baseline.
(3) Clean the probe tip (see
Maintenance) and then run
Sensor Check.
(4) See Principles of
Operation.
(1) Empty the low
reflectance standard and
refill with distilled water
prior to each usage.
(2) Clean the probe tip.
See Maintenance.
(3) At the end of the
Sensor Check procedure
update the sensor
coefficients. See
Verification of Sensor
Performance.
(1) See Setting up for a
Bioreactor Run: Step 2.
(2) Set the sensor
averaging time constant to
the highest value allowed
by the growth rate of the
culture. See step 5A of
Setting Up and
Configuring.
Suggested Remedies
This message is provided for
informational purposes. No action
is required.
“User cal error”
Calibration transform is set to
Log-Log, and negative or
zero-valued data encountered.
Base Unit Trouble-Shooting
Observation
Possible Causes
Analog output is not
(1) Range setting is not
responsive to changes in optimal.
sensor readings.
(2) Analog output power is
not plugged in.
BE3000 Software Communication Trouble-Shooting
Observation
Possible Causes
BE3000 instrument not
(1) Probe or USB cable not
detected by BE3000
connected.
Virtual Instrument
(2) USB driver was not
software.
correctly installed.
(3) BE3000 device not
recognized by a USB hub.
Computer
(1) The computer went into
communication with the
sleep mode.
BE3000 device was
(2) Power to the BE3000
interrupted.
device was interrupted.
(3) Connecting through a
USB hub device.
(1) Make sure the baseline is
correctly set. AND
(2) Wait for the biomass to
increase above 0. OR
(3) Switch to the linear transform
method. See Editing, Generating,
and Saving a Calibration.
Suggested Remedies
(1) Make sure the AO range
setting matches with the maximum
anticipated biomass reading. See
Analog Output
(2) Plug in the power. See step 3
of Connecting the Probe and
Base Unit
Suggested Remedies
(1) See Connecting the Probe and
Base Unit.
(2) See USB Driver TroubleShooting at the end of this table.
(3) Re-boot your computer.
(1) See Step 1 of Setting Up and
Configuring.
(2) Consider connecting the
computer to a power source that
can provide uninterrupted power
(e.g. battery back-up).
(3) If possible, connect your
BE3000 device directly to a USB
port on your computer, rather than
connecting through a USB hub
(which we have found to be less
reliable).
USB Driver Trouble-Shooting
If you are connecting the BE3000 instrument to a computer via USB, but the instrument
76
is not recognized by the User Interface software, the USB driver software may not have
been installed correctly. The following procedure describes how to check and, if
necessary, re-install the USB communication driver software. This procedures assumes
that you have already installed the User Interface software (if not, first follow the steps in
Software Installation).
1)
2)
3)
Check to make sure that the driver was installed properly by checking the status in
Device Manager:
a. Make sure that probe and base unit are connected and that the base unit is
connected to a USB port on your computer.
b. Press the windows Start button and choose Control Panel.
c. Locate and double-click on the “Device Manager” icon.
d. Scroll down the devices to “Ports (COM & LPT)” and view the listed devices
(by clicking on the windows expansion arrow).
e. One of the listed ports should be “BugLab BE3x00 (COMx)”, where x is an
integer (e.g. “BugLab BE3x00 (COM4)”). If no such device is listed, skip to
step 3 below.
f.
Right-click on the USB Serial Port and select “Properties”.
g. Click on the “General” tab. The “Device status” window should be displaying
“This device is working properly”. If not, skip to step 2 below.
h. Click on the “Driver” tab. The driver that is listed should be BugLab LLC
version 1.3.1.0, or higher. If it is not, proceed to step 2 below.
If the device is not working properly or the wrong driver version is listed, update
the driver software:
a. Under the “Driver” tab, select “Update Drive”.
b. In the Update Driver Software window that pops up, click on “Browse my
computer for driver software” and hit “OK”.
c. Browse to the Windows\INF folder in your root drive (e.g. C:\Windows\INF).
Choose “Let me pick from a list of device drivers on my computer”. Click on
“BugLab BE3x00” and then “Next”.
Start up the BE3000 Virtual Instrument software and wait for the list of detected
BE3000 devices to finish updating
END
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